salmonella enterica by live cell imaging and ... filenovel approaches to understand the...
TRANSCRIPT
Novel approaches to understand the intracellular lifestyle of
Salmonella enterica by live cell imaging and ultrastructural
studies
Neue Ansätze zum Verständnis der Interaktion intrazellulärer Salmonella
enterica und Wirtszellen mittels Lebendzell-Mikroskopie und ultrastruktureller
Untersuchungen
Der Naturwissenschaftlichen Facultät
der Friedrich-Alexander-Universität-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer.nat.
Vorgelegt von
Roopa Rajashekar
Bangalore, India
2
Als Dissertation genehmigt von der Naturwissen-
schaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 29.07.2010
Vorsitzender der
Promotionskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. Michael Hensel
Zweitberichterstatter: Prof. Dr. Andreas Burkovski
3
This work is dedicated to my beloved mother
Late Smt. Padma Rajashekar
4
Table of contents
1 INTRODUCTION 6
1.1 Salmonella and salmonellosis 6
1.2 Epidemiology of Salmonella infection 6
1.2.1 Sources of infection 7
1.2.2 Medically relevant representatives 7
1.2.3 Salmonella pathogenesis and disease outcome 8
1.3 Secretion systems in Salmonella 11
1.3.1 SPI1 and SPI2 Type Three Secretion Systems 11
1.3.2 SPI1 Type Three Secretion Systems 13
1.3.3 SPI2 Type Three Secretion Systems 13
1.3.4 Cross-Talk between SPI1 and SPI2 Type Three Secretion Systems 14
1.4 Pathogenicity Islands of Salmonella enterica 14
1.4.1 Salmonella Pathogenicity Island 1 (SPI1) 15
1.4.2 Salmonella Pathogenicity Island 2 (SPI2) 15
1.5 Endocytosis 17
1.5.1 The Endocytic Pathway 18
1.5.2 Features of Phagolysosome 19
1.5.3 Involvement of SNAREs and Rabs in phagolysosomal biogenesis 19
1.5.4 Involvement of phosphoinositides in the endocytic pathway 21
1.5.5 The dynamic role of cytoskeleton in phagosome maturation 22
1.6 Intracellular lifestyle of Salmonella 23
1.6.1 Divergent lifestyles of intracellular pathogens 23
1.6.2 Salmonella as a facultative intracellular pathogen 25
1.6.3 SCV- The intracellular habitat of Salmonella 26
1.6.4 Avoidance of host-derived antimicrobial radicals 29
1.6.5 Virulence factors and their molelcular mechanism in controlling the intracellular fate of Salmonella 29
1.6.6 Effectors proteins of the SPI2-T3SS and their contribution to intracellular life and Salmonella induced phenotypes 30
2 RATIONALE AND AIMS OF THE PROJECT 34
3 RESULTS AND PUBLICATIONS 36
5
3.1 Dynamic Remodeling of the Endosomal System during Formation of Salmonella-Induced Filametns by Intracellular Salmonella enterica 36
3.2 Novel functions of SPI2 effector proteins during intracellular pathogenesis of Salmonella enterica revealed by live cell and Ultrastructural analyses 72
3.3 Ultrastructural analysis of SCV and Biogenesis of SIF by Electron tomography 98
4 DICUSSION 116
5 SUMMARY 129
6 LIST OF PUBLICATIONS AND CONTRIBUTION OF CO-AUTHORS 133
7 REFERENCES 134
8 ABBREVIATIONS 146
9 CURRICULUM VITAE 149
10 ACKNOWLEDGEMENTS 152
6
1 INTRODUCTION
1.1 Salmonella and salmonellosis
Salmonella is a Gram-negative, rod-shaped, peritrichous flagellated and motile
enterobacterium. It is approximately 0.7 to 1.5 µm in diameter and 2 to 5 µm in length (Fig
1). Salmonella are facultative anaerobes, mostly found in contaminated water (sewage water)
and in packed food products like poultry, meat, eggs and milk products which are under-
processed. Salmonella are closely related to the Escherichia genus and widely distributed in
animals and humans. Salmonellacauses food-borne illnesses such as typhoid and paratyphoid
fever which are systemic infections associated with septicemia. However gastroenteritis is a
more self limiting condition.
Figure 1: Morphology of Salmonella. Salmonella enterica Typhimurium (red) is a rod shape bacteria with
peritrichous flagella. (Adopted from http://www.textbookofbacteriology.net/themicrobialworld/S.enterica.jpeg).
1.2 Epidemiology of Salmonella infection
Outbreaks of Salmonella infections mainly typhoid fever caused by S. enterica serovar
Typhi and gastroenteritis caused by S. enterica serovar Typhimurium are quite common
globally both in developed and developing countries. There is usually outbreak of
gastroenteritis when humans or animals ingest contaminated food or water. It has been
estimated that there are approximately 20 million cases of human illness every year due to
typhoid resulting in about 200,000 deaths worldwide (Crump et al., 2004).
Salmonella enterica
Peritrichous flagella
7
In Germany, more than 80% of the human isolates from the cases reported to the Enteric
Reference Centre at the Robert Koch Institute in 1995 were comprised of serovar Enteritidis
(61.3%) and serovar Typhimurium (23.4%) (Rabsch et al., 2001). Intermittent shedding of the
pathogen by domestic animals is thought to provide a constant reservoir for infection and
contamination of food. Industrialization and large scale food distribution, increased
consumption of raw or slightly cooked foods, an increase in immuno-compromised patient
populations, deteriorated public infrastructure and evolution of multi-drug-resistant
Salmonella have been all proposed as possible reasons for the steady increase in the incidence
of Salmonella infections (Darwin & Miller, 1999). Therefore more stringent quality control
measures are required to prevent these outbreaks.
1.2.1 Sources of infection
Contamination of ground water due to its mixing with sewage water is the common
source of Salmonella where bacteria can survive for several weeks. Aquatic vertebrates,
notably birds and reptiles, are important vectors of Salmonella. Poultry, cattle, and sheep
frequently being agents of contamination, Salmonella can be found in food, particularly meats
and raw eggs.
1.2.2 Medically relevant representatives
S. enterica serovar Choleraesuis (Bacillus paratyphoid B and C), is an intestinal
commensal in pigs, humans can be infected by ingesting sick animals, the bacteria
causes septicemic Salmonellosis in swine.
S. enterica serovar Paratyphi
o S. Paratyphi A, solely a human pathogen, causes paratyphoid A, transmission
by contact and contaminated food or water.
o S. Paratyphi B, in central Europe usually a human pathogen, causes
paratyphoid B; transmission by contact and contaminated food, water or fly
excrement.
S. enterica serovar Typhi, causes systemic infection called typhoid fever in humans.
The source of infection is usually contaminated food or water. 3–5 % of all patients
remain permanent carriers of the pathogen.
8
S. enterica serovar Typhimurium (also referred to as S. typhimurium), causes a wide
range of infections in birds and mammals ranging from self limiting gastroenteritis to
severe systemic paratyphoid diseases; conveyed by contaminated food.
1.2.3 Salmonella pathogenesis and disease outcome
Entry of Salmonella to its animal host is a huge challenge as it encounters a series of
unique environments, such as temperature, pH, osmolarity, and nutrient availability. Bacteria
also encounters host innate immunity like phagocytes which could engulf and kill the
bacteria. Pathogens sense these changes and adapts to the environment by coordinated
programs of gene expression that provides an adaptive advantage in each new host
environment. In order to adapt to such conditions pathogens activates specific virulence
mechanisms that allow them to resist, evade, or even systematically manipulate the innate
immunity.
As Salmonella is a Gram-negative bacterium it possess antigen repertoire such as
lipopolysaccharide and lipoproteins of the outer membrane and the host innate immune
system detects the presence of microbial pathogens using receptors that recognize these
structures (Medzhitov & Janeway, 2000). A host response is stimulated by interaction of these
microbial signature molecules with specific host receptors at the intestinal mucosa. Bacterial
pathogens that survive the innate immune effectors may persist in the host, which allows
recognition of microbial signature molecules which in turn activate cytokine production and
inflammation. All these persistent host responses lead to disease outcome.
The clinical symptoms associated with Salmonella infection are enteric (typhoid) fever
and gastroenteritis (Miller & Pegues, 2000). Enteric fever is a systemic illness that results
from infection with the exclusively human pathogens, Salmonella enterica serovar Typhi and
Paratyphi. Clinical symptoms include pain in abdomen, diarrhea, headache, fever etc. The
pathological hallmark of enteric fever is mononuclear cell infiltration in the intestinal Peyer's
patches, mesenteric lymph nodes, spleen, and bone marrow. Many non-typhoidal Salmonella
strains, such as S. enteritidis and S. typhimurium, cause self-limited enteritis in humans. As
the stomach environment is acidic due to gastric juices with high pH, Salmonellae exhibit an
adaptive acid-tolerance response on exposure to low pH (Garcia-del Portillo et al., 1993). In
the stomach, the bacterium comes in contact with the intestinal mucosal layer before
encountering and adhering to cells of the intestinal epithelium.
9
Salmonellae express several fimbriae that contribute to their ability to adhere to intestinal
epithelial cells (Baumler et al., 1996).
Microscopic studies have revealed that Salmonellae invade epithelial cells by a
morphologically distinct process termed as bacterial-mediated endocytosis (Francis et al.,
1992). This unique process is characterized by membrane ruffles formation and is different
from receptor-mediated endocytosis. Shortly after bacteria adhere to the apical epithelial
surface, profound cytoskeletal rearrangements occur in the host cell, disrupting the normal
epithelial brush border and inducing the subsequent formation of membrane ruffles that reach
out and enclose adherent bacteria in large vesicles (Fig 2). This process is different from the
receptor-mediated endocytosis. Following bacterial internalization, Salmonella resides in a
host-derived vacuole called as Salmonella-containing vacuole (SCV) which transacts to the
basolateral membrane and the apical epithelial brush border reconstitutes.
Fig 2: Invasion of Salmonella and membrane ruffle formation in epithelial cells: Scanning electron micrograph
showing Salmonella.typhimurium entering a Hep-2 cell through bacterial mediated endocytosis. Membrane
ruffles extend from the cell surface, enclosing and internalizing adherent bacteria. (Adopted from Ohl & Miller,
2001).
10
As studied in mice models, Salmonellaappear to preferentially adhere to and enter the
microfold cell (M cells) of the intestinal epithelium, although invasion of normally
nonphagocytic enterocytes also occurs (Jones et al, 1994). M cells (Fig 3) are specialized
epithelial cells that sample intestinal antigens through pinocytosis and transport these antigens
to lymphoid cells that underlie the epithelium in Peyer's patches (Brandtzaeg et al., 1989).
This activity is important in the priming of mucosal immunity. In bovine epithelium,
however, Salmonella do not appear to interact preferentially with M cells and the relative role
of M cells and enterocyte invasion in different animal hosts is not well understood.
Salmonellamay also passively cross the intestinal epithelial barrier following phagocytosis by
migrating CD18-positive phagocytes (Vazquez-Torres et al., 1999). Furthermore, many SPI1
encoded genes have also been implicated in mediating macrophage apoptosis in vitro (Chen et
al., 1996) and loss of electrolytes and fluid secretion, contributing to enteritis and intestinal
inflammation (Wallis & Galyov et al., 2000).
Fig 3: Diagrammatic representation of pathogenesis of Salmonella in the human gut: Orally ingested
Salmonellae survive at the low pH of the stomach and evade the multiple defenses of the small intestine in order
to gain access to the epithelium. Salmonellae preferentially enter M cells, which transport them to the lymphoid
cells (T and B) in the underlying Peyer's patches. Once across the epithelium, Salmonella serotypes that are
associated with systemic illness enter intestinal macrophages and disseminate throughout the reticuloendothelial
system. By contrast, non-typhoidal Salmonella strains induce an early local inflammatory response, which
results in the infiltration of PMNs (polymorphonuclear leukocytes) into the intestinal lumen and causes diarrhea.
(Adopted from Haraga et al., 2008).
11
In addition to invasion of the intestinal epithelial barrier, Salmonella serotypes clinically
associated with enteritis induce a secretory response in the intestinal epithelium and initiate
recruitment and transmigration of neutrophils into the intestinal lumen (Galyov et al., 1997).
Once across the intestinal epithelium, Salmonella serotypes are then taken up by macrophages
which then cause systemic infections. Salmonella employs several virulence factors to
overcome the harsh environment within the macrophages, survive, replicate and further
disseminate into deeper tissues. (Alpuche-Aranda et al., 1994).
1.3 Secretion systems in Salmonella
Gram-negative bacterial species have to secrete the virulence factors across several
barriers like the periplasmic space, outer membrane and phospholipid bilayer of inner
membrane. To efficiently secrete virulence factors, Gram-negative bacteria have evolved
several types of secretion system. Based on this, secretion systems have been grouped as Type
I to Type VI secretion system. In this section more emphasis will be given to the Type III
secretion system, as this is the key for Salmonella pathogenesis.
1.3.1 SPI1 and SPI2 Type Three Secretion Systems
General Properties of Type III Secretion Systems
Type III secretion systems (further referred to as T3SS) are specialized bacterial
apparatus whose central function is the delivery of bacterial proteins into eukaryotic cells
(Cornelis & Van Gijsegem, 2000; Galan & Collmer, 1999; Hueck et al., 1998). These systems
are related to flagella export system during the course of evolution. T3SS are among the most
complex protein secretion systems known in bacteria and is composed of more than 20
proteins. T3SS also known as molecular syringes, are highly regulated, both at the
transcriptional and post-transcriptional level (Lucas & Lee, 2000). Such regulation is essential
for the functioning of the secreted proteins at the site of delivery in a coordinated manner.
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Fig 4: Salmonella Type Three Secretion System Needle Complex: a) Electron micrographs of negatively stained
isolated needle complexes. b) Cross-section of the structure of the needle complex indicating the location of its
different substructures. c) Surface rendering of the structure of the needle complex. Shown here are different
views of the structure of a 20-fold complex with 20-fold symmetry imposed. (Adopted from Galan et al., 2006)
The main feature of the secretion apparatus is the formation of the needle complex
formed by a subset of structural components (Kubori et al., 1998). This structure spans both
the inner and outer membranes of the bacterial envelope and resembles the flagellar hook
basal body complex. The base of the needle complex of the Salmonella T3SSs is composed of
three proteins, InvG, PrgH, and PrgK (Kimbrough & Miller, 2000; Kubori et al., 1998 &
2000). The assembly of the needle complex occurs in a step-wise fashion that is initiated by
the sec-dependent secretion of the base components, which are assembled into a multi-ring
structure (Sukhan et al., 2001). Proteins that are transported through T3SSs carry multiple
signals that route them to the secretion apparatus and eventually to the host cell (Cheng &
Schneewind, 2000; Cornelis & Gijsegem, 2000). It has been demonstrated that there is a built-
in hierarchy in the type III secretion process (Kubori et al., 2000). As discussed below,
spectrum of effector proteins ultimately delivered by T3SS into host cells includes a variety of
enzymes capable of modulating cellular functions.
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1.3.2 SPI1 Type Three Secretion Systems
The SPI-1 T3SS (Fig 4) is present in all Salmonella serovars. Four proteins, SpaO,
InvJ, PrgI and PrgJ, are a part of the complex of other proteins involved in protein secretion
and/or the assembly of the needle complex (Collazo & Galan, 1996; Collazo et al., 1995;
Kimbrough & Miller, 2000; Kubori et al. 1998 & 2000). Three proteins, SipB, SipC, and
SipD, are required for protein translocation across the eukaryotic cell membrane (Collazo &
Galan, 1997). The remaining proteins that carry out effector functions within the host cell are
therefore known as effector proteins. It is becoming increasingly clear that different effector
proteins modulate different cellular processes and therefore are most likely involved in
various stages of bacterial infection. The SPI-1 T3SS continues to be active after bacterial
internalization and once intracellular the bacteria can deliver virulence proteins into the host
cell cytosol through the membrane of the enclosing phagosome (Collazo & Galan, 1997).
Therefore, it could be suggested that Salmonella reprograms its SPI-1 T3SS machinery to
change the battery of proteins delivered to the host cell. Such reprogramming may occur as a
consequence of changes in the pattern of effector-protein gene expression or through
posttranscriptional regulatory mechanisms.
1.3.3 SPI2 Type Three Secretion Systems
The SPI2 T3SS was identified by the signature taged mutagenesis approach (Hensel et
al., 1995; Shea et al., 1996). The locus was independently identified by sequence analysis
comparing Salmonella enterica and E. coli K12 genomes (Ochman et al., 1996), and it was
found that a set of sequences including the SPI2 locus were present exclusively in S. enterica
but absent from E. coli K12.
Genetic analysis has revealed that SPI2 T3SS is among the most distantly related
systems to the SPI1 T3SS. A subset of the putative secreted proteins (e.g., SseB, SseC, and
SseD) (Fig 5) are believed to be required for effector protein translocation into the host cell
based on their amino acid sequence similarity to proteins that carry out this function in other
T3SSs. Putative effector proteins include SpiC, SifA, SseF, SseG, PipB2, etc. (Beuzon et al.,
2000; Guy et al., 2000; Uchiya et al., 1999). A subset of SPI2 T3SS substrates possess
approximately 100-amino acid amino-terminal domain that shares striking sequence similarity
(Beuzon et al., 2000; Guy et al., 2000; Miao et al., 1999). It has been proposed that this
domain contains signals required for secretion and targeting for the SPI2 T3SS.
The host of effector proteins and their phenotypes will be discussed later in the intracellular
lifestyle of Salmonella section.
14
Fig 5: SPI2 encoded needle complex: Detection of SPI-2-encoded needle complex using SPI2 encoded proteins
by immunoelectron microscopy. S. typhimurium wild type was grown overnight in PCN-P media at pH 5.8 and
cells were processed for immuno-EM. Fixed bacteria were incubated with antisera against SseB: (Adopted from
Chakravortty et al., 2005)
1.3.4 Cross-Talk between SPI1 and SPI2 Type Three Secretion Systems
There is evidence indicating that the SPI1 and SPI2 T3SSs work independently. In
general, mutations affecting the function of one system do not significantly affect the
phenotypes mediated by the other system. In addition, SPI1 is expressed initially during the
invasion of the host cell and SPI2 is expressed at the later stage of infection when Salmonella
is in the intracellular compartment. However the effector protein, SspH1, has been shown to
be the target of both the secretion systems. Further, SopD has been shown to be secreted
through the SPI1 T3SS (Jones et al., 1998) while it also exhibits structural features, at its
amino terminus, similar to those present in SPI2 secreted proteins (Miao et al., 1999). In
addition, certain mutations in SPI2 influence the expression of SPI1 genes (Deiwick et al.,
1998) suggesting the existence of a mechanism that coordinates the temporal expression
patterns of these two systems.
1.4 Pathogenicity Islands of Salmonella enterica
Salmonella pathogenicity islands also called as ‘SPI’ are clusters of genes on
pathogenicity islands that harbor virulence traits including host cell invasion and intracellular
pathogenesis. At least 12 pathogenicity islands have been described so far. In this section a
15
brief account of pathogenicity islands 1 and 2 will be discussed as it is central for invasion
and intracellular pathogenesis.
1.4.1 Salmonella Pathogenicity Island 1 (SPI1)
SPI1 is a 40kb region located at locus 63 on Salmonella chromosome (Mills et al.,
1995; Hensel et al., 2004) that encodes the SPI1 type III secretion systems (Fig 6). Type III
secretion system is responsible for the secretion of complex set of effector proteins that is
required for Salmonella invasion. One subset of proteins mediates the invasion of non-
phagocytic cells and the second subset is involved in the enteropathogenesis.
During the process of infection, SPI1 effecter proteins induce bacterial mediated
endocytosis directing host cell actin cytoskeleton rearrangements by interfering with the host
signal transduction pathways (Galan & Zhou, 2001). Three effector proteins SopE, SopE2,
and SopB/SigD stimulate actin polymerization by different mechanisms and have partially
redundant functions. Host Rho GTPases (CDC42 and Rac) is activated by 3 proteins that
directly bind to N-WASP and WAVE complexes, which inturn leads to activation of the
Arp2/3 complex leading to actin polymerization. Actin dynamics is influenced by SipA which
acts by decreasing critical concentration for actin polymerization, inhibiting depolymerization
of actin filaments and increasing the bundling activity of T-plastin and stability of the actin
bundles. SipC also promotes nucleation and thereby bundling of actins. After completion of
internalization, the proteins SptP and SigD are involved in restoring the normal architecture of
the infected cell. While SigD destabilizes the actin filament attachment to the membrane, the
tyrosine phosphatase activity of SptP inhibits CDC42 and Rac resulting in release of free G-
actin monomers.
1.4.2 Salmonella Pathogenicity Island 2 (SPI2)
The SPI2 is a 40kb locus (Fig 6) adjacent to the valV tRNA gene (Hensel et al., 1997).
This region encodes a second Type III secretion system in Salmonella that plays an important
role in intracellular pathogenesis. Both SPI1 and SPI2 encoded T3SS are the result from
independent events of horizontal gene transfer (Hensel et al., 1997; Foultier et al., 2002). S.
typhimurium deficient in SPI2 encoded T3SS are highly attenuated in murine Salmonellosis
(Shea et al., 1996). The SPI2 system is under the control of regulatory system SsrAB. It has
also been shown that global regulator PhoPQ and OmpR/EnvZ is also controlling SPI2 gene
expression. (Lee et al., 2000).
16
Fig 6: Organization of SPI1 and SPI2 pathogenicity island of S.typhimurium: Several genes in SPI1 and SPI2
have different designations: sprA = hilC = sirC; sipBCDA = sspBCDA; ssaB = spiC; ssaC = spiA; ssaD = sipB;
ssrA = spiR. The functional classes of SPI1 and SPI2 genes are represented by different colors (Adopted from
Hasen Wester et al., 2001)
There are a set of about 22 effector proteins that are secreted by the SPI2 system
(Andrea et al., 2007). Most of the SPI2 proteins are secreted within the phagosome and are
important for bacterial replication and associated pathogenesis.
Several intracellular pathogens and Salmonella in particular have evolved mechanisms
to use a variety of virulence traits to invade the host and cause infection. How pathogens
mimic the basic cellular functions and exploit this to their advantage is of prominent interest.
Therefore, to understand how these changes are brought about in the host, it is important to
know the basic cellular machinery required for host functions, like the endocytosis, vesicular
trafficking, protein synthesis etc. The following section gives an overview of some of the
common cellular functions required for the normal functioning of the cell.
17
1.5 Endocytosis
Endocytosis is a process by which cells absorb molecules (such as proteins, fluids)
from external environment by engulfing them with their cell membrane.
There are 4 types of endocytotic uptake namely:
a) Clathrin-mediated endocytosis
b) Caveolae
c) Macropinocytosis
d) Phagocytosis
Clathrin-mediated endocytosis: The process is mediated by small (approx. 100 nm in
diameter) vesicles whose coat is made up of a complex of proteins that mainly associated with
the cytosolic protein clathrin. Clathrin-coated vesicles (CCVs) are found in virtually all cells
and from domains of the plasma membrane termed clathrin-coated pits. Coated pits can
concentrate large extracellular molecules that have different receptors responsible for the
receptor-mediated endocytosis of ligands, e.g. low density lipoprotein, transferrin, growth
factors, antibodies and many others.
Caveolae: The surface of many cell types exhibit non-clathrin coated plasma membrane buds,
called as caveolae. They are made up of the cholesterol-binding protein caveolin (Vip21) with
a bilayer enriched in cholesterol and glycolipids. Caveolae are small (approx. 50 nm in
diameter) and constitute up to a third of the plasma membrane area of the cells. They are
abundantly found in smooth muscle, fibroblasts, adipocytes, and endothelial cells (Parton et
al., 2007).
Macropinocytosis: The highly ruffled regions of the plasma membrane forms invagination
like a pocket, which then pinches off into the cell to form a vesicle (0.5–5 µm in diameter)
filled with large volume of extracellular fluid and this process is referred to as
macropinocytosis. The filling of the pocket occurs in a non-specific manner. The vesicle then
travels into the cytosol and fuses with other vesicles such as endosomes and lysosomes
(Falcone et al., 2006).
Phagocytosis: The process by which cells internalize particulate matter larger (0.75 µm in
diameter), for example small-sized dust particles, cell debris, micro-organisms, apoptotic cells
18
etc is called phagocytosis. Phagocytosis restricted to certain specialized cells called as
macrophages, dendritic cells.
These processes involve the uptake of larger membrane areas than clathrin-mediated
endocytosis and caveolae pathway. However, off late there has been a more generalized
classification of endocytosis based on clathrin-dependent or independent pathways
(Lundmark et al., 2008).
1.5.1 The Endocytic Pathway
The endocytic pathway is the mechanical maturation of the endocytic vesicle (where the
cells have engulfed extracellular material by endocytosis as described above). This early
vesicle formed soon after uptake of external material are called as the early endosomes or also
referred to as phagosomes. The fate of early endosome depends on the route of maturation it
takes during the endocytic pathway. Following formation, endocytic vesicles are targeted to
early endosomes or phagosomes and recycling endosomes. Recycling endosomes are
morphologically and biochemically distinct from early endosomes. They are often in a
juxtanuclear location, near the microtubule-organizing centre, are less acidic (pH 6.5) than the
early endosome or phagosome, and can be identified by the presence of Rab11 (Lemmon et
al., 2000).
Early endosomes or phagosomes (formed nearly 30min post endocytosis) are organelles
which are often tubulovesicular, and can be typically recognized by presence of markers like
Rab5 or early endosome antigen 1 (EEA1). The lumen of early endosomes is relatively poor
in proteases and is mildly acidic, with a pH of >6.0. Alternatively, molecules destined for
degradation progress from sorting to late endosomes (Fig 7). Late endosomes are more acidic
than early endosomes, with a pH of 5.5, and are enriched in hydrolytic enzymes. They are
multivesicular nature, and can be identified by the presence intracellular markers like Rab7,
Rab9, lysobisphosphatidic acid, mannose-6-phosphate receptor and lysosomal-associated
membrane proteins (LAMPs) (Mukherjee et al., 1997).
The final step of phagosome maturation in the endocytic cycle is the fusion of
phagosome to lysosome to from phagolysosome. These organelles contain the bulk of active
proteases and lipases, and are extremely acidic (pH<5.5). Although the characteristic feature
of lysosomes is the presence of LAMPs and hydrolytic enzymes such as cathepsin D these
markers are also present on late endosomes. Phagosomes rapidly lose the characteristics of
early endosomes, while attaining those of late endosomes.
19
1.5.2 Features of Phagolysosome
Phagolysosome becomes enriched in late-endosomal components like Rab7, the
mannose-6-phosphate receptor and lysobisphosphatidic acid (Fig 8). Phagolysosomes are also
characterized by presence of hydrolytic proteases, such as processed cathepsin D, and by the
acquisition of an extremely acidic pH, reported to be as low as 4.5 (Jahraus et al., 1998).Due
to several experimental evidences it can be proposed that phagosomes direct their own
maturation which is supported by further experiments showing that phagosomes isolated 1 h
after formation fuse preferentially with lysosomes, but not with earlier endocytic organelles
(Desjardins et al., 1997).
There are several models suggesting the fusion of Phagosome and lysosome, and one
such model is the `kiss and-run' model for phagosome maturation (Duclos et al., 2000).
According to this, phagosomes undergo only a transient and partial fusion with endocytic
organelles which can be called as “kiss”, this allows the transfer of selected membrane and
luminal contents between the phagosome and the endosome, followed by a fission event
called “run”, preventing the complete intermixing of the two compartments.
1.5.3 Involvement of SNAREs and Rabs in phagolysosomal biogenesis
Vesicles are targeted by SNAREs where SNARE is a soluble N-ethylmaleimide-
sensitive factor-attachment protein receptor, a family of membrane-tethered coiled-coil
proteins, N-ethylmaleimide- sensitive factor (NSF) and NSF-attachment proteins (SNAPs)
which are critical determinants of vesicular transport in a variety of systems (Gotte &
Mollard, 1998). The hallmark of SNAREs is that they contain conserved heptad repeat
sequences in their membrane-proximal regions that form coiled coil structures. These families
of SNAREs are required for vesicle docking and fusion. Small GTPases of the Rab family are
the next most important family of proteins implicated in vesicle trafficking. They are proteins
which by utilization of ATP, undergoes phosphorylation of GTP to GDP inturn carrying out
function. They have restricted organellar distribution and because they can promote the
selective tethering of vesicles with target organelles (Zerial & McBride, 2001). Several Rab
GTPases localize to the endocytic pathway for e.g. Rab5 associates predominantly with the
sorting endosome, Rab4 and Rab11 locate preferentially to recycling endosomes, while Rab7
is localized to late endosomes and lysosomes (Bucci et al., 2000).
20
Fig 7: Diagrammatic representation of sequential events in endocytic pathway: The cartoon shows the schematic
representation of the endocytic pathway. (Adopted from Drecktrah et al., 2007)
Rab5 is the best characterized of the endosomal Rab proteins. It has many functions
such as formation of clathrin-coated vesicles (Bucci et al., 1992), the tethering and fusion of
coated vesicles with sorting endosomes, homotypic fusion between sorting endosomes
(Gorvel et al., 1991) and the movement of sorting endosomes along microtubules (Nielsen et
al., 1999). In addition, Rab proteins are involved with late endosomes like for example Rab7
is localized to late endosomes. Data obtained with dominant-negative and constitutive active
Rab7 mutants have indicated that this GTPase are important in the regulation of late-
endocytic traffic (Feng et al., 1995).
21
It has been suggested that Rab7 could regulate the transition from early to late endosomes
(Vitelli et al., 1997).
Fig 8: Phagosome Biogenesis: The above cartoon shows the schematic biogenesis of phagosome to
phagolysosome in the endocytic pathway. (Adapted from Drecktrah et al., 2007)
Although, many other Rab proteins have been found on the phagosomal membrane such as,
Rab3, Rab4, Rab5, Rab7, Rab9, Rab10, Rab11 and Rab14 (Desjardins et al., 1994), very little
information is available regarding their function.
1.5.4 Involvement of phosphoinositides in the endocytic pathway
PI 3-kinases are a family of enzymes that phosphorylate the D-3 hydroxy group of
phosphoinositides. Products of phosphatidylinositol 3-kinases (PI 3-kinases) are thought to
play a role in the traffic of membranes along the endocytic pathway and are also critical for
phagosome maturation. Class I PI 3-kinases are heterodimeric enzymes composed of catalytic
and adaptor or regulatory subunits. Thus PI 3-kinases regulate endocytic traffic. In
mammalian cells, it has been shown that inhibitors of PI 3-kinase such as wortmannin and
LY294002 affect endocytic traffic (Backer et al., 2000)
22
1.5.5 The dynamic role of cytoskeleton in phagosome maturation
Microtubules
Microtubules are one of the key cytoskeletal elements required for various functions in
host that include cell division, cell movement and also in intracellular trafficking.
Microtubules are long filaments made up of protein subunits (mainly and tubulins) which
also possess the polarity and orientation of the tubules. Microtubules provide polarized tracks
(their minus and plus ends are oriented towards the centre and periphery of the cell
respectively) that guide organellar dynamics, which in turn is driven by a variety of molecular
motors. Most important among these motors are members of the dynein and kinesin families,
which propel organelles in a centripetal or centrifugal direction respectively. Microtubule-
associated proteins serve as adaptors between microtubules, the motor proteins and their cargo
(Pierre et al., 1992).
Various components of the endocytic pathway are positioned in particular domains of
the cell in a microtubule-dependent fashion, and their location influences the endosomal
progression. Traffic between endosomes and lysosomes is severely affected when
microtubules are disrupted using pharmacological agents like nocodazole (which is
microtubule depolymerizing agent (Matteoni & Kreis, 1987). Similarly, interference with the
proper functioning of kinesin or dynein is known to impair the fusion between early and late
endosomes (Burkhardt et al., 1997). In similar lines, phagosomes were shown to possess
bidirectional motility along microtubules although displacement towards the minus ends
predominated (Blocker et al., 1997) over the plus end movement. Reconstitution assays using
cell-free systems identified kinesin as the motor that powers the plus-end-directed movement
of phagosomes, and kinectin, which is transmembrane receptor for kinesins, acts as an
adaptor. In contrast, minus-end oriented motility is brought about by dyneins, coupled through
the dynactin complex (Blocker et al., 1997).
Despite intense studies, the mechanism of how phagosomes become linked to
microtubules remains unclear. Members of the Rab family could play a major role in bridging
vesicles to microtubules. For example it was found that Rab4a bound to a cytoplasmic dynein
chain (Bielli et al., 2001), Rabphilin11 an effector of Rab11, was shown to align along
peripheral microtubules, and Rab5 was demonstrated to regulate endosome motility on
microtubules (Mammoto et al., 1999). Thus Rabs are known to associate with early
phagosomes and could play a role in microtubule-dependent centripetal motion.
23
All these above constituents of the cellular trafficking system are important for the cell to
maintain homeostasis between its external and internal environment. These systems are
effectively exploited by various microorganisms to create their own niche and bring about
pathogenesis in host cells.
Pathogenic microorganisms (bacteria, viruses or protozoa), have evolved mechanisms
to disrupt and utilize host cellular machinery for the outcome of the disease. In this case how
Salmonella takes advantage of the host intracellular trafficking for its survival is discussed
below in detail.
1.6 Intracellular lifestyle of Salmonella
Salmonella enterica belongs to the group of bacterial pathogens that have evolved a
facultative intracellular lifestyle. This remarkable ability allows Salmonella to grow in the
environment, extracellularly inside a host organism, and inside eukaryotic cells where certain
cell types permit intracellular replication of the pathogen. Salmonella has the ability to invade
non-phagocytic eukaryotic cells by injection of effector proteins through a T3SS system
(Schlumberger & Hardt, 2006). A more conventional route of uptake is phagocytosis that does
not require an active contribution of the bacteria. After internalization, Salmonella resides in a
membrane-bound compartment, or parasitophorous vacuole, which is maintained throughout
intracellular life. A large number of bacterial factors are required for successful intracellular
pathogenesis. Recent observations have shown that the intracellular pathogen can actively
manipulate the host cell in order to maintain the parasitophorous vacuole, to avoid
antimicrobial activities of the host cell and to acquire nutrients for intracellular replication. In
this section, the intracellular lifestyle of Salmonella, the manipulations that alter normal
functions of the host cell and the molecular function of virulence factors of Salmonella will be
described in detail. Some light is also thrown on the current frontiers in our understanding of
the intracellular lifestyle of Salmonella and discuss how modern approaches such as live cell
imaging and tomographic electron microscopy may contribute to extend these frontiers.
1.6.1 Divergent lifestyles of intracellular pathogens
A facultative intracellular lifestyle is a common observation among pathogenic
bacteria. It is widely considered that intracellular life on one hand protects bacteria from a
variety of humoral immune responses of the host organisms and, on the other hand, allows the
utilization of host cell molecules that may otherwise be limited in the host organisms.
24
However, the benefits of intracellular life appear less prominent if one considers the activation
of the mechanisms of host immune defense against intracellular organisms and the restricted
access to nutrients for bacteria that reside within a membrane-bound compartment.
Salmonella has evolved efficient strategies to cope with these challenges.
Bacterial pathogens have evolved two main concepts of intracellular life. Members of
one subgroup have the ability to lyse the parasitophorous vacuole and escape into the host cell
cytoplasm. These bacteria usually also show actin-based intracellular motility which allows
bacteria to infect neighboring cells in a tissue without the need to re-enter the extracellular
space. Listeria monocytogenes and Shigella spp. are the best-studied pathogens in this group,
and recent additions include Rickettsia typhi, Burkholderia pseudomallei and Mycobacterium
marinum (reviewed in Gouin et al., 2005). An obvious benefit of the intra-cytoplasmic
lifestyle of bacteria is the direct access to metabolites and various nutrients of the host cell.
One important disadvantage is the recognition by the immune system due to the presentation
of foreign cytosolic proteins by major histocompatibility complex class I (MHC-I) and the
cytotoxic killing of infected cells. Recent studies showed that intracellular motility not only
enables bacteria to spread intercellularly, but also provides an important contribution to
prevention of tagging by the ubiquitin system and proteasomal degradation (Birmingham &
Brumell, 2006; Perrin et al., 2004).
The other, larger, subgroup of intracellular bacteria has evolved various strategies to
persist and replicate within membrane-bound compartments or 'pathogen-inhabited
compartments' (PIC), and Salmonella belongs to this group. Bacteria that inhabit PICs are
often are also invasive. However, an intracellular lifestyle within a PIC can also occur after
passive uptake of bacteria by phagocytosis. One example is Mycobacterium tuberculosis that
has no strategy for invasion, but is phagocytosed by cells of the host immune system, is able
to prevent killing by host cells due to the unique structure of the cell envelope, and transforms
its parasitophorous vacuole into a replication-permissive PIC.
The number of intracellular strategies probably equals the number of pathogens
inhabiting PICs. Recent molecular and cellular analyses show that bacteria in PICs use highly
evolved and rather divergent strategies to modify their host cells and to create their
intracellular niches. At first glance; one can characterize the PIC of a given pathogen in order
to understand the specific mechanisms. For example, Legionella pneumophila is a natural
intracellular pathogen in amoebae, but can also infect the human lung if transmitted by
25
contaminated aerosols from air condition or hot water devices (reviewed in Vogel & Isberg,
1999).
This pathogen generates a unique PIC that is characterized by the presence of markers
characteristic for the endoplasmic reticulum of the host cell (Kagan & Roy, 2002). Other
bacteria such as M. tuberculosis appear to modify the normal maturation of a phagosome in an
early stage, resulting in a PIC that is characterized by the absence of the canonical markers of
late endosomes or lysosomes. Further individual strategies can be observed for the formation
of the PIC of pathogens such as Chlamydia spp., Coxiella spp., Bartonella spp., Francisella
spp., and many others, and there are excellent recent reviews for more details (Schaible &
Haas, 2009). Although the formation of a PIC is a well-known virulence trait of S. enterica,
the biogenesis of this compartment and the mechanisms are still topic of many current
investigations.
1.6.2 Salmonella as a facultative intracellular pathogen
The ability of Salmonella enterica to infect host cells and to survive and proliferate
within a PIC has been observed soon after the discovery of this species as an infectious agent.
Early observations revealed the role of intracellular life for the ability of Salmonella to cause
systemic infections (Fields et al., 1986). Salmonella is able to infect various eukaryotic host
cells. The bacteria are invasive and can deploy the SPI1-encoded T3SS to initiate complex
modifications of the host, resulting in a process termed macropinocytosis that ultimately leads
to the uptake. Salmonella can proliferate in epithelial cells and non-activated macrophages.
The survival in activated macrophages and persistence in dendritic cells have been observed,
while the situation in fibroblasts appears to more diverse – here, a restriction of replication
was seen (Garcia-del Portillo et al., 2008).
Entry into host cells can either occur via bacteria-mediated invasion, or by
phagocytosis. The bacteria are thought to primarily replicate in macrophages, as they are
found in the lymphatic tissues and organs during systemic infection. Salmonella strains that
are defective for macrophage replication are avirulent in mouse models of infection, which
underscores the importance of bacterial survival and replication in macrophages for disease
outcome (Fierer & Guiney, 2001). The ways of entry into the cell probably affects the initial
phase of maturation of the compartment containing the bacterium. However, Salmonella
internalized by either invasion or phagocytosis both induce the formation of a specialized PIC
that is referred to as ‘Salmonella-containing vacuole’ or SCV. The SCV has certain
26
characteristics of a late endosomal compartment, such as the presence of lysosomal
glycoproteins and the acidic luminal pH.
1.6.3 SCV- The intracellular habitat of Salmonella
Similar to the intracellular pathogens introduced earlier on, Salmonella is able to
actively modify the biogenesis of a novel membrane-bound compartment in which the
bacteria can survive and efficiently replicate (Fig 9). Although this compartment has some
features in common with late endosomes, other properties are unique and may be the result of
a fine-tuned interference with normal host cell functions. In the following, the features of this
compartment will be described, termed 'Salmonella-containing vacuole' or SCV.
Salmonella is capable of infecting and replicating in many different cell types. Unlike
non-phagocytic cells, Salmonella can enter macrophages by several endocytic processes,
including SPI1-induced macropinocytosis (Ishibashi & Arai, 1995; Oh & Straubinger, 1996).
Following SPI1-induced macropinocytosis, Salmonella remains inside the intracellular SCV
and persistence of proliferation may occur. Within the SCV, the bacteria can persist
intracellularly for hours to days, making it a unique phagosome with respect to the normal
progression of phagolysosomal maturation and recycling. Although there has been some
controversy within the field, several reports have shown that Salmonella can survive within
macrophages in which the lysosomal compartments have fused with the SCV (Ishibashi &
Arai, 1995; Oh & Straubinger, 1996). Consequently, the avoidance of phagolysosomal fusion
is unlikely to be a major pathogenic strategy of Salmonella. Studies in various cell types have
also demonstrated that the vacuole acidifies, however, depending on the mechanism of host
cell entry, vacuolar acidification may be delayed in both macrophages and epithelial cells
(Drecktrah et al., 2006; Rathman et al., 1996). The SCV undergoes similar maturation steps
like the phagosome as discussed in (section 1.6.6). Furthermore, cholesterol has been reported
to accumulate in the membrane of the SCV (Catron et al., 2002).
The presence or absence of different markers on the persistent SCV may simply
indicate that they are variably detected rather than reflect whether or not the SCV has matured
through a normal endocytic pathway. It has been suggested that the SCV can acidify and fuse
with lysosomes. The ability of Salmonella to survive exposure to lysosomal contents is
mediated by its resistance to antimicrobial peptides, nitric oxide and oxidative killing -
important features for its survival within macrophages and to virulence (Chakravortty &
Hensel, 2003; Vazquez-Torres & Fang, 2001). This is supported by the observation that
Salmonella spp. mutants that are sensitive to these compounds are attenuated in virulence in
27
the murine model, whereas knock-out mice deficient for the production of these compounds
have increased susceptibility to Salmonella spp. (Chakravortty & Hensel, 2003; Vazquez-
Torres & Fang, 2001). Salmonella sense the acidic environment of the SCV, resulting in the
induction of various regulatory systems that promote intracellular survival, for example, by
surface remodeling of the protein, carbohydrate and phospholipid components of the bacterial
envelope (Bijlsma & Groisman, 2003). Such regulatory systems include OmpR/EnvZ,
PhoP/PhoQ, RpoS/RpoE, PmrA/PmrB, Cya/Cyp and cyclic diGMP, all of which confer
resistance to antimicrobial peptides and oxidative stress (Groisman & Mouslim, 2006). The
phagosomal environment is acidic, with a pH range of <5 to 5.5, has a concentration of
magnesium and calcium in the 1 mM range and contains antimicrobial peptides, oxygen and
nitrogen radicals that can damage the bacterial cell (Prost et al., 2007).
Various studies indicate that both pH and antimicrobial peptides are important
signatures of the phagosomal environment and such conditions activate many of the
regulators that are implicated in Salmonella virulence (reviewed in Haraga et al., 2008). It is
likely that several sensory systems respond to the phagosome environment and cooperate to
orchestrate the complex cascade of events that are necessary to alter the bacterial surface and
promote intracellular survival (Groisman & Mouslim, 2006). The best characterized of these
is the PhoQ sensor, which promotes resistance to antimicrobial peptides (Shi et al., 2004).
PhoQ contains a novel acidic domain that is bridged to the bacterial inner membrane by
interactions with metal ions and binds to and initiates responses to antimicrobial peptides
(Prost & Miller, 2008). It also responds to pH by structural changes that are determined by
regions of the protein separate from the metal ion bridges involved in antimicrobial peptide
sensing (Bader et al., 2005; Cho et al., 2006). After phagocytosis, Salmonella undergoes
extensive bacterial surface remodeling, as has been shown for the lipid A component of
lipopolysaccharide (LPS) during growth within macrophages (Prost & Miller, 2008).
28
Fig 9: Biogenesis of Salmonella containing vacuole in the endocytic pathway: Shown is the invasion and
intracellular replication of Salmonella, with maturation of SCV by interaction of various markers. (Fig adopted
from chapter written by Rajashekar & Hensel for Caister Academic Press, Titled - Salmonella: from Genome to
Function)
Bacterial molecules that the host can recognize as indicators of infection, such as the
SPI1 T3SS and flagellin, are repressed and the LPS structure is altered (Trent et al., 2001).
Some of the specific surface modifications include a decrease in length of the O antigen,
which is the repeating carbohydrate polymer of the LPS, alterations to the number of acyl
chains in the structure of the lipid A component of LPS, and changes in the protein content of
the outer membrane, the inner membrane and the peptidoglycan layer. Synthesis of enzymes
that allow the bacteria to cope with oxidative and nitrogenous radicals also occurs. Micro
array studies have shown that up to 919 S. enterica serovar Typhimurium genes are
differentially regulated in response to the phagosomal environment, demonstrating that
dramatic transcriptional and post-translational changes probably occur when Salmonella
29
makes the transition from a nutrient-rich extracellular environment to the intracellular
environment (Eriksson et al., 2003).
1.6.4 Avoidance of host-derived antimicrobial radicals
During the intracellular life, Salmonella is exposed to a variety of antimicrobial
defense mechanisms that would ultimately result in the killing and degradation of intracellular
bacteria. The nature and efficiency of the antimicrobial mechanisms vary between the cell
types infected by Salmonella. While activated macrophages can mount a large panel of highly
reactive radicals and active degradative enzymatic activities, this capacity is less pronounced
in resting macrophages, while epithelial cells are only able to activate a limited number of
antimicrobial activities.
In phagocytic cells, the main anti-microbial activity is attributed to the action of
reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) that are
generated by the constitutively expressed NADPH oxidase (phox) and the inducible nitric
oxide synthase (iNOS), respectively. Both ROI and RNI are short-lived, highly reactive
molecules with the ability to damage various macromolecules of the internalized microbes. If
both species of reactive intermediates are produced simultaneously, the reaction product
peroxynitrate is formed that has an even higher capacity to kill microbes. The role of phox and
iNOS in the control of Salmonella proliferation has been demonstrated on the cellular level as
well as in infection studies in murine models. Being confronted with these potent defense
mechanisms, intracellular Salmonella have to mount efficient counteractions. These include
activation of repair mechanisms to compensate the radical-induced damages, detoxification of
radicals by expression of catalytic enzymes but also avoidance of exposure to radicals by re-
directing the radical-producing enzymes away from the SCV (Chakravortty et al., 2002).
1.6.5 Virulence factors and their molelcular mechanism in controlling the
intracellular fate of Salmonella
Once inside the SCV, Salmonella sense the novel environment and as a result, the
expression and assembly of the SPI2-encoded T3SS is induced (Cirillo et al., 1998). Although
the function of this T3SS in pathogenesis requires further molecular understanding, it has
been shown to be essential for virulence in the mouse model of infection (Shea et al., 1996).
Mutants deficient in the SPI2-T3SS cannot replicate efficiently in tissue-culture cells and are
highly attenuated in animal models of infection. It is likely that the role of this T3SS during
disease is to promote intracellular replication within cells of the intestine during the acute
30
phase of the infection and in other organs during the persistent state. Current hypotheses for
the function of the SPI2-T3SS propose the promotion of intracellular replication by altering
host vesicular trafficking, so that useful metabolic molecules, such as amino acids and lipids,
are routed to the SCV and the vesicular compartment membrane is expanded. The SPI2-
encoded T3SS is active in intracellular Salmonella and the translocation of a large set of 20
and probably more effector proteins into host cells has been observed (recent overview in
Haraga et al., 2008). Salmonella mutant strains defective in the SPI2-T3SS and thus unable to
translocate the cocktail of effector proteins are highly attenuated in systemic pathogenesis
and, on the cellular level, show a highly reduced intracellular survival and proliferation. Yet,
these mutant strains maintain an intracellular habitat that is similar to the SCV formed by
wild-type Salmonella.
1.6.6 Effectors proteins of the SPI2-T3SS and their contribution to
intracellular life and Salmonella induced phenotypes
To date, at least 20 Salmonella spp. effector proteins are known to be translocated by
the SPI2-T3SS across the phagosomal membrane into the cytoplasm of the eukaryotic cell
(Haraga et al., 2008). However, their specific roles in promoting intracellular replication or
modifying vesicular movement are not yet understood (for a list of SPI2-T3SS effectors, their
functions and binding partners, see (Haraga et al., 2008). In addition, no individual
translocated effector has definitively been shown to alter vesicular trafficking. Although it has
been reported that SpiC alters endosome fusion in vitro (Uchiya et al., 1999) and binds to
proteins that are implicated in vesicular trafficking (Lee et al., 2002; Shotland et al., 2003), its
role as a T3SS effector protein remains controversial (Freeman et al., 2002; Yu et al., 2002).
As SpiC has not been universally observed to be translocated into eukaryotic cells, and is
required for the translocation of several, if not all, SPI2-T3SS effectors as well as surface
expression of translocon proteins, SpiC is more likely part of the SPI2 secretion apparatus
rather than an effector (Yu et al., 2002).
The most important translocated effectors, by virtue of causing virulence defects when
mutated, are SifA, SseF, SseG, SopD2, SseJ, and PipB2 (Garcia-del Portillo et al., 1993;
Henry et al., 2006; Hensel et al., 1998; Jiang et al., 2004; Ohlson et al., 2005). The
observation that S. enterica serovar Typhimurium that lacks any single SPI2 T3SS effector
protein cannot cause the same virulence attenuation in mice as a mutant strain that lacks the
entire SPI2 T3SS suggests that many effectors function cooperatively to exert their effects on
31
the host cell. Furthermore, the deletion or mutation of many effector genes has no virulence
phenotype, which implies that their functions might be redundant. Early studies of SPI2 T3SS
effectors, which primarily focused on determining their subcellular localization in mammalian
cells, revealed that they might have specific targeting sequences that direct localization to
endosomal compartments (Kuhle & Hensel, 2002), the Golgi apparatus (Salcedo & Holden,
2003), the actin cytoskeleton (Miao et al., 2003) and the microtubule network (Kuhle et al.,
2004).
This indicates that components of these host-cell structures might be the intracellular
targets of the SPI2 T3SS. Among the various effector proteins, SifA probably has the most
prominent role (Beuzon et al., 2000). Mutant strains defective in sifA fail to generate
Salmonella-induced filaments (SIF), and loss of the SCV and escape into the host cell
cytoplasm has been observed. The contribution of SifA to Salmonella virulence and
intracellular replication has been demonstrated by in vitro as well as by in vivo studies. A sifA
strain is highly attenuated in a murine model and reduced in the replication in cultured cells.
Furthermore, a sifA strain escapes into the cytoplasm of infected host cells (Beuzon et al.,
2000) where its fate is variable depending on the host cell type. The sifA strain cannot grow in
macrophages as it is killed by the highly stringent environment of the macrophage cytoplasm
(Beuzon et al., 2002; Brumell et al., 2001). In contrast, the same mutant replicates very
rapidly in the cytoplasm of epithelial cells, and high bacterial proliferation kills the host cell
too rapidly to allow efficient net replication in an infected cell population. This observation is
of interest in relation to previous studies by the group of W. Goebel that used microinjection
of intracellular pathogens into the cytoplasm of various host cells (Goetz et al., 2001). Defects
in cytosolic replication were observed for microinjected Salmonella but not for Listeria and
these results were considered an indication of the specific adaptation to the nutritional
conditions of intracellular pathogens with cytosolic or vacuolar lifestyle.
Regardless of the mechanism of SIF formation, the filaments may function to increase
the size of the SCV to accommodate the growing numbers of Salmonella during intracellular
replication (Beuzon et al., 2000). SIF formation is dependent on SifA (Beuzon et al., 2000;
Brumell et al., 2001b; Stein et al., 1996), but also, to a lesser extent, on SseF, SseG (Guy et
al., 2000; Kuhle & Hensel, 2002), SopD2 (Jiang et al., 2004) and PipB2 (Knodler & Steele-
Mortimer, 2005). However, it may also be modulated by other effectors. SifA was recently
shown to bind to the host cell protein SKIP (SifA and kinesin interacting protein) (Boucrot et
al., 2005). The same authors also found that, if SKIP was depleted by RNA interference in S.
enterica serovar Typhimurium-infected cells, the cells failed to form SIF, suggesting that SIF
32
formation requires SKIP. In addition, SifA is a member of a family of T3SS-effector proteins
that possess the motif WxxxE (tryptophan (W)-variable (x)-xx- glutamate), and for some of
these effectors a function in mimicking small GTPases of the host cell was demonstrated
(Alto et al., 2006). SifA, similar to GTPases, contains a carboxy-terminal Caax (cysteine (C)-
aliphatic residue (a)-a-x) motif that is prenylated and S‑ acylated by host-cell enzymes
(Boucrot et al., 2003), it is possible that SifA uses a GTPase-type mechanism for membrane
localization and, perhaps, SIF formation (Jackson et al., 2008). Furthermore, the region of
SKIP that interacts with SifA is a pleckstrin homology domain, which is commonly found in
proteins that bind to signaling lipids and other regulatory molecules, including those that
modulate Rho GTPases.
The roles of PipB2, SopD2, SseF and SseG in SIF formation are not entirely clear.
Salmonella strains that lack any of these effectors do not induce SIF as efficiently as the wild-
type strain (Guy et al., 2000; Kuhle & Hensel, 2002). Instead, infection of cultured cells with
these mutants tends to lead to the formation of ‘pseudo-SIF’, which extend from the SCV and
co-localize with LAMP-1, but do not contain effectors. This suggests that in the absence of
these proteins, the ability to form SIF, which is defined as being entirely LAMP-1-positive, is
impaired. Furthermore, induction of SIF involves multiple steps that are orchestrated by
different effectors. Transient expression of PipB2 in mammalian cells induces the movement
of LAMP-1-positive compartments to the cell periphery, which is probably the result of its
interaction with the plus-end-directed microtubule motor kinesin (Knodler & Steele-
Mortimer, 2005). This activity might contribute to the outward extension of the SCV, which
would promote SIF formation. SopD2, which has homology to the SPI1-T3SS translocated
effector SopD, has also been shown to cooperate with SifA to induce SIF, but by an as yet
unidentified mechanism (Brumell et al., 2003). SseF and SseG promote the aggregation of
endosomal vesicles and recruit Golgi-derived exocytic vesicles to the SCV, which suggests
that Salmonella is able to usurp both endocytic and exocytic cellular transport processes
(Kuhle et al., 2006; Salcedo & Holden, 2005). However, it is not known how these activities
directly influence SIF formation. The deletion of SseJ and SpvB can cause an increase in SIF
formation at later time points during the infection of cultured cells, which suggests that these
proteins have SIF down-regulatory functions.
SpvB is an actin-specific ADP-ribosyltransferase that promotes actin depolymerization
(Tezcan-Merdol et al., 2005). SseJ has homology to glycerophospholipid cholesterol acyl
transferase enzymes, which can remove acyl chains from phospholipids and transfer them to
cholesterol in a two-step deacylase-acyltranferase reaction (Nawabi et al., 2008). In fact,
33
purified SseJ has deacylase activity in vitro and has been shown to be required for its
virulence in mice (Ohlson et al., 2005).
It has been proposed that SseJ and SifA have complementary roles in maintaining the
integrity of the SCV membrane, because sifA-deficient S. typhimurium tends to lose the SCV
membrane but does not do so if SseJ is also lacking (Ruiz-Albert et al., 2002). This suggests
that, in the absence of SifA, SseJ could cause damage to the SCV by its enzymatic activity.
Nawabi and coworkers suggest that SseJ has phospholipase activity in vivo and that this
function could be localized to the endosomal membrane, resulting in cleavage of
phospholipids from the SCV membrane (Nawabi et al., 2008). The cleavage of acyl chains
from phospholipids could then promote curvature of the membrane or produce discrete lipid
environments that serve as platforms for promoting vesicle fusion and binding scaffolding
proteins and such activity could modulate SIF formation. However, how these functions and
SIF formation in general, are important for Salmonella-induced disease is currently not
understood. It is plausible that SIF increase the size of the SCV in a specific and directional
fashion that promotes bacterial replication and/or redirect nutrient- rich organelles to the SCV.
In this regard, the SPI2 T3SS could help Salmonella to replicate inside the phagosome and
gain important nutrients for growth, yet avoid some of the host-defense mechanisms and
inflammatory responses that would result from their release into the cytoplasm.
34
2 RATIONALE AND AIMS OF THE PROJECT
Host endosomal pathway involves endocytic and exocytic systems important for the
normal cellular trafficking. Intracellular pathogens like Salmonella take advantage of host
endosomal system to establish infection. Previously, several intracellular phenotypes of
Salmonella have been defined using fixed cells and Salmonella has been proposed to remodel
or hijack the host cellular machinery. Salmonella enterica proliferate within a specialized
membrane compartment, the Salmonella-containing vacuole (SCV), and interfere with the
microtubule cytoskeleton and cellular transport. We addressed several previously described
phenotypes of Salmonella including Salmonella induced filaments (Garcia-del Portillo et al.,
1993), loss of SCV membrane due to lack of effector protein SifA (Beuzon et al., 2000) etc. In
addition, from the earlier work in our group, we have reported several novel phenotypes
including “Pseudo SIF”, microtubule bundling (Kuhle et al., 2002), and redirection of
exocytotic transport vesicles to the SCV (Kuhle et al., 2006) by virulent Salmonella was
reported. SIF were described only in HeLa cells and their occurrence in other natural cell
types such as macrophages (RAW or peritoneal macrophages) or (bone marrow derived)
dendritic cells was not known. Further, there was no exact information on the dynamics and
biogenesis of SIF. All the above mentioned phenotypes were described using fixed cells.
Fixation of biological samples might disrupt or destroy the fine membrane structures and
molecular interaction which could otherwise cause artifacts and wrong interpretation of the
results. Since cellular trafficking is a dynamic event, fixing the samples hamper the
visualization of important cellular events
Therefore, unlike the previous studies, which were mainly based on the fixed cell setup,
we wanted to develop and use live cell imaging assays to study vesicular trafficking with
particular relevance to SCV and SIF formation in more detail and its effects on the outcome of
Salmonella infection. The basic scientific question was to study the aspect of endosomal
remodeling by Salmonella in real time as these are spontaneous and dynamic events involving
host vesicular trafficking. Therefore the main objectives of the project were to:
I) Establish live cells assays to track Salmonella infection in living HeLa cell culture
model.
II) Use the live cell assays to determine in detail SIF formation taking place in real
time. Is SIF formation confined to HeLa cells or a more general phenotype?
35
III) Investigate the contribution of individual SPI2 effector proteins in SIF formation
and biogenesis of SCV.
IV) Detailed analysis of SCV and SIF organization at the ultrastructural level and
uncover the underlying aspects that could lead us to understand SIF biogenesis.
The understanding of exciting results that emerged from all these studies paved way for
greater insight into Salmonella lifestyle with its host in real time.
36
3 RESULTS AND PUBLICATIONS
Chapter 1
3.1 Dynamic Remodeling of the Endosomal System during Formation of Salmonella-
Induced Filametns by Intracellular Salmonella enterica
Roopa Rajashekar1, David Liebl
2, Arne Seitz
2 and Michael Hensel
1,*
Mikrobiologisches Institut, Universitätsklinikum Erlangen, Wasserturmstr. 3-5, D-91054 Erlangen, Germany 2EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany
*Corresponding author: Michael Hensel, [email protected] Note: This work was published in the journal Traffic. 2008 Dec; 9(12):2100-16. Epub 2008
Sep 19
Abstract
The infection by Salmonella enterica results in the massive remodeling of the
endosomal system of eukaryotic host cells. One unique consequence is the formation of long
tubular endosomal compartments, so-called Salmonella-induced filaments or SIF. Formation
of SIF requires the function of type III secretion system and is a requirement of efficient
intracellular proliferation of Salmonella. Using high resolution live cell imaging approaches
and electron microscopy, we report for the first time the highly dynamic characteristics of SIF
and their ultrastructural properties. In the early phase of infection (4 - 5 h), SIF display highly
dynamic properties in various types of host cells. SIF extend, branch and contract rapidly and
a stabilized network of SIF is formed later ( 8 h after infection). The velocities of SIF
extension and contraction in the different phase of infection were quantified. Our observations
lead to novel models for the modification of host cell transport processes by virulence factors
of intracellular Salmonella.
Introduction
The transport of vesicles in eukaryotic cells is a sophisticated process that requires the
coordinated function of cytoskeletal transport routes, motor proteins and regulators (reviewed
in 1, 2, and 3). For example, endosomal vesicles containing internalized material undergo a
canonical series of maturation steps that involve the directional transport on microtubules (4).
37
The modification of this cellular machinery is of vital importance of intracellular pathogens.
Salmonella enterica is a Gram-negative bacterium able to invade non-phagocytic cells, but
also to survive after internalization by phagocytic cells (recent overview in 5). Regardless of
the way of uptake, Salmonella remains located within a membrane-bound compartment
termed Salmonella-containing vacuole (SCV). The SCV has special features such as the
presence of late endosomal/lysosomal membrane proteins (e.g. LAMP-1), an acidic pH and
the juxtaposition to the trans-Golgi network (TGN) microtubule-organizing center and
nucleus in epithelial cells. To control its intracellular fate, Salmonella actively manipulates the
host cell and a type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2
(SPI2) is essential for this pathogenic lifestyle (reviewed in 6). The SPI2-T3SS is induced
inside the SCV and translocates a set of 19 and possibly more effector proteins across the
vacuolar membrane. While the initial steps of the biogenesis of the SCV do not appear
dependent on the SPI2-T3SS, its function is later required to maintain the integrity of the SCV
and its specific subcellular localization, to prevent the delivery of antimicrobial host factors to
the SCV, to modify the organization of the host cell cytoskeleton, and to alter vesicular
transport.
A remarkable phenotype induced by intracellular Salmonella is the formation of so-
called Salmonella-induced filaments or SIF (7). SIF are tubular membrane structures that
contain various late endosomal/lysosomal markers that are also characteristic for the SCV.
The formation of SIF has been characterized in epithelial cell lines and SIF were shown to
form along microtubules (8, 9). Previous work indicated that the induction of SIF formation is
dependent on the function of the SPI2-T3SS (10). Mutant strains defective in the SPI2-T3SS
and unable to translocate effector proteins do not induce SIF formation. More specifically, a
subset of SPI2 effector proteins is involved in SIF formation consisting of SifA, SseF, SseG,
SopD2 and PipB2 (10-14). Recent studies demonstrated the involvement of microtubule
motor proteins in SIF formation (15, 16). A model has been proposed in that the balanced
activity of opposing motor proteins dynein and kinesin is required for the maintenance of the
SCV in perinuclear localization and the effective intracellular replication of Salmonella (17,
18).
Despite these observations the biogenesis of SIF is not well understood and it is not
clear how SIF formation correlate to the intracellular survival and proliferation of Salmonella.
The involvement of host cell transport processes prompted us to investigate the dynamics of
the host cell endosomal system in response to Salmonella infection using live cell approaches
38
and ultrastructural analyses. Our observations indicate that SIF formation is induced in
various cell types infected with Salmonella and that SIF are highly dynamic structures.
Live cell setups for endosomal remodeling by Salmonella
Endosomal remodeling and formation of SIF has been reported previously for
Salmonella-infected epithelial cells (7) and it is now clear that this phenotype requires the
function of the SPI2-T3SS and a subset of SPI2-T3SS effector proteins (10).So far, these
studies have been performed with fixed cells that were analyzed for the extent of SIF
formation, the presence of specific host cell markers, and bacterial factors involved. For a
more detailed understanding of SIF biogenesis, we developed live cell setups and investigated
the dynamics of SIF formation.
A characteristic feature of the SCV as well as of SIF is the presence of highly abundant
lysosomal glycoproteins such as LAMP-1. We transfected HeLa cells with a vector expressing
LAMP-1-GFP, followed by infection with S. typhimurium that constitutively express GFP or
mCherry. SIF formation was observed in live host cells infected with S. typhimurium WT, but
not in cells infected with strains defective in the SPI2-T3SS (ssaV) or a strain deleted in sifA,
encoding the key effector for SIF induction (sifA) (Fig. 1A). Due to the uniform size and
shape of the intracellular bacteria, the bacterial GFP fluorescence in general was easily
distinguishable from the GFP label on endosomal membranes. Infected cells were also fixed
and immuno-stained for LAMP-1. SIF formation was observed in LAMP-1-GFP transfected
and non-transfected cells. A nearly complete colocalization of LAMP-1-GFP and endogenous
LAMP-1 was confirmed in fixed and immuno-stained cells (Suppl. Materials, Fig. S 2). The
frequency of SIF-positive cells and the gross morphological appearance of SIF were similar in
transfected and non-transfected HeLa cells (data not shown). Furthermore, the intracellular
proliferation of Salmonella was not altered by LAMP-1-GFP transfection. These results
indicated that LAMP-1-GFP is a useful marker for further analyses.
As alternative markers, we investigated the loading of host cells after infection with
fluid phase markers such as Alexa568-dextran (Fig. 1 BC) or 10 nm gold particles conjugated
with BSA rhodamine (Fig. 1D). Labeling of the SCV and of SIF was observed with either
tracer.
39
Fig. 1. Tracing of the endosomal system in living host cells infected with Salmonella. A) HeLa cells were
transfected with LAMP-1-GFP (green) and infected with S. typhimurium wild type (WT), ssaV- or sifA-deficient
strains expressing GFP (green). Living cells were imaged 5 h after infection using a with Zeiss Axiovert 200M
wide field microscope. B) HeLa cells were infected with S. typhimurium WT expressing GFP (green) and the
fluid phase marker Alexa568-dextran was added 5 h after infection. Living cells were imaged 2 h after addition
of the marker. C) HeLa cells were transfected as in (A) and infected with WT Salmonella expressing mCherry,
but in addition Alexa568-dextran was added as in B) at 5 h after infection. Note the strong co-localization of
both markers in most of the tubular compartments. D) HeLa cells were infected with WT Salmonella and the
fluid phase marker 10 nM gold-BSA-rhodamine was added 5 h after infection. After 2 h, the cells were washed
and immediately imaged. Note the SCV that shows colocalization between intracellular Salmonella and gold-
BSA-rhodamine (arrowhead). Scale bars, 5 and 2.5m for overview and insert, respectively.
40
Both approaches were combined and the analyses of living cells indicated that infection with
Salmonella WT resulted in the formation of SIF that were positive for LAMP-1-GFP and
Alexa568-dextran added 5 h after infection with Salmonella (Fig. 1C). The majority of SIF
were positive for both markers, although some SIF were only labeled with the fluid tracer or
LAMP-1-GFP. Note that LAMP-1-GFP labels the cytoplasmic face of the SIF membrane,
while Alexa568-dextran labels the lumen of the SIF tubules. Experiments were performed
with fluid tracers added 5 h after infection and imaging was performed 2 h after addition of
the tracer. Interestingly, we observed that in a portion of infected cells analyzed, the SCV as
well as SIF were labeled with the tracer. The entire lumen of the SCV appeared positive for
the tracer (see Fig. 1D). These observations are in accord with the recently reported
continuous interaction of the SCV with the endosomal system (19). Because of its higher
photostability, we preferentially used LAMP-1-GFP for further investigations in live cell
experiments with HeLa cells.
Intracellular Salmonella alter the overall endosomal organization
Using the live cell setup, we followed the intracellular fate of Salmonella and the
organization of the labeled endosomes over time. We observed the fusion of LAMP-1-GFP-
positive vesicles with SCV as early as 2 h after infection ( Fig. 2A, Movie 1). As expected,
the formation of SIF was observed in cells infected with Salmonella WT but not in cells
infected with SPI2-deficient Salmonella. The formation of tubular extensions positive for
LAMP-1-GFP emerging from the SCV was observed as early as 3 h after infection and these
tubules were rapidly changing in size ( Fig. 2B, Movie 2).
In accord with previous studies (7, 20) we noticed that the number of filaments
increased over time, resulting in a complex meshwork of filaments visible after 10 h of
infection with Salmonella. During this process, the increase of SIF was correlated with
decrease of late endosomal/lysosomal vesicles ( Fig. 2C). The numbers of LAMP-1-GFP-
positive vesicles in living cells were determined under various infection conditions ( Fig.
2D). We counted the total number of LAMP-1-GFP-positive, spherical structures per infected
cell as indicated in the supplementary materials (Fig. S 3). In non-infected cells or cells
infected with a SPI2 strain the number of vesicles was not different from WT-infected cells
observed 3-6 h after infection. In contrast, a highly decreased number of LAMP-1-GFP-
positive vesicles were observed in cells infected for 8-9 h with WT Salmonella. These
observations indicate that LAMP-1-GFP-containing membranes are reorganized into tubular
SIF by the action of intracellular Salmonella.
41
Fig. 2. Salmonella-induced changes to the host cell endosomal system. HeLa cells were transfected with a vector
for LAMP-1-GFP expression and infected with Salmonella WT or SPI2-deficient strains expressing GFP.
Imaging was done using a Perkin Elmer UltraView RS spinning disc confocal microscope. A) The fate of
LAMP-1-GFP-positive vesicles (arrowhead) in the vicinity of the SCV (arrow) was followed 2 h after infection.
Tracking of individual vesicles showed the fusion of these vesicles to the SCV (corresponding to Suppl. Movie
1). B) Emergence of tubular structures from SCV 3 h after infection (Suppl. Movie 2). Note the rapid growth of
tubular structure (indicated by arrowheads) from an existing tubular compartment. C) And D) Overall
distribution of LAMP-1-GFP-positive membranes at early and late time point of infection. C) Representative
mock-infected cells or cells infected with WT or SPI2-deficient strain are shown 4 h or 9 h after infection as
indicated. Projections of 5 Z sections with 0.5m spacing are shown. Scale bars, 5 m. D) The overall number of
LAMP-1-GFP-positive vesicles was determined in living HeLa cells at various times points after mock infection
or infection with WT or SPI2 strains. The mean number ( standard deviation) of LAMP-1-GFP-positive
spherical vesicles was determined for 20 cells per experimental condition and the data shown are representative
for three independent experiments. Scale bar, 5m
42
Ultrastructural features of SIF
In all previous studies, the alterations of the endosomal system caused by Salmonella
were analyzed using immunofluorescence microscopy techniques. Here we applied electron
microscopy (EM) to investigate structural features of SIF with higher resolution. To correlate
immunofluorescence microscopy to EM, we processed cells infected with Salmonella WT for
immunogold labeling on cryosections. We found abundant labeling of the LAMP-1 on SCV
as well as on membranes of adjacent vesicles Fig 3A. In a portion of the sections analyzed for
LAMP-1-GFP expressing cells tubular membrane structures with frequent anti GFP-labeling
were observed (Fig 3B).
To reveal the spatial distribution of the SIF we further analyzed vertical ultrathin
sections from conventional (plastic) flat-embedded samples to better preserve the orientation
of adherent cells. We observed specific tubular membrane structures in Salmonella-infected
cells that did not appear in mock-infected control cells (Fig. S 4) or cells infected with the
SPI2 strain (data not shown). These membrane tubules were similar in proportion to
prolonged tubular mitochondria but clearly distinguishable by lack of internal membrane
cristae. Transmission EM analyses indicated typical diameters of SIF in the range of 160 nm
(± 39 nm) and we propose that these structures correspond to SIF observed in living cells or
fixed immuno-stained cells. For some tubular membranes, a clear continuity between the SCV
and the lumen of the SCV was observed as shown in Fig 3C and D and occasionally, multiple
membrane structures were observed for SIF and the SCV.
Many tubular membranes were also found without connection to the SCV. This
observation may be interpreted as cross-section through the three-dimensional structure since
ultrathin sections represents merely 1% of the total cell volume. However, free SIF without
connection to SCV were also observed by live imaging. Interestingly, closer inspection of the
vicinity of the SCV (Fig. 3C) and the tip of SIF (Fig. 3D) indicated the presence of large
amounts of small spherical as well as tubular vesicles. These might represent a pool of
vesicles that interact with SCV and SIF and could contribute to the increase of SCV diameter
and SIF length. However, since the fate of a vesicle cannot be followed by EM, it is also
possible that there vesicles result from fission from the SCV or SIF.
43
Salmonella induces highly dynamic tubular endosomal aggregates
We next followed the formation of SIF in living HeLa cells after transfection with
LAMP-1-GFP and infection with GFP-expressing Salmonella. Time lapse series were
acquired from 1 h to 10 h after infection and a representative example is shown in Fig 4
(Movie S3). We observed that the replication of intracellular bacteria was detectable starting
at 3.5 h and larger clusters of bacteria or micro-colonies were observed from 6 h onwards. We
also noted the formation of tubular LAMP-1-GFP-positive structures as early as 3 h after
infection. The number and the extent of these tubular structures increased over time and based
on the appearance of the tubular structures we considered them to be SIF. Closer analyses of
the time lapse movies indicated that individual SIF were highly dynamic with respect to
extension but also contraction, branching or fusion with other SIF (Movie 3). We noted that
these dynamics are most prominent shortly after induction of SIF.
The velocity of growth and collapse or contraction, as well as the frequency of
branching and merging of SIF decreased over time, but SIF number and length increased. At
the end of the observation period, a complex network of SIF was established similar to the
appearance of SIF in fixed cells. At this time point, the SIF were almost static and showed
only little alteration in length. These novel observations for a unique cellular structure
induced by pathogenic bacteria prompted us to characterize SIF formation in living cells in
more detail and in a quantitative manner.
Dynamic SIF are formed in various host cells
Previous studies investigated SIF in epithelial cell lines such as HeLa. As the
observation of SIF in cell lines raises some concerns about the broader biological relevance of
the phenomenon, we set out to investigate SIF formation in other cell lines as well as in
primary cells. One previous report showed the appearance of SIF-like tubular membranes in
IFN stimulated macrophages infected with Salmonella (21). IFN stimulation results in the
activation of macrophages that coincides with changes in the gross cell morphology, most
importantly the spreading of the cell and tight adherence to a substrate.
44
Fig. 3. Ultrastructure of Salmonella-containing vacuoles and SIF. HeLa cells were infected with Salmonella WT
and cells were processed for EM 10 to 12 h after infection as described in experimental procedures. A) and B)
Cells transfected with LAMP-1-GFP were infected with Salmonella WT and immunolabeled for LAMP-1 (A) or
GFP (B), processed for cryosections and immunogold-labeled for GFP. Scale bars, 200nm. C) and D) Infected
cells were embedded in EPON resin and processed for transmission EM. Representative SCV in connection with
SIF are shown. Boxes in lower magnification panels indicate the positions of high magnification panels. C) In
addition to the SCV connected to a SIF, a large number of small globular vesicles are present in the vicinity of
the SCV (arrowheads). Scale bars, 1 m (left panel) and 200 nm (right panel). D) Numerous small vesicles are
present at the tip of SIF connected to an SCV (arrowheads). Scale bars, 1 m (left panel) and 200 nm (middle
and right panels).
45
The murine macrophage-like cell line RAW264.7 was IFN stimulated, pulse-chased with
BSA coupled to 10 nm gold-rhodamine (Fig. S 5A) or Alexa568-dextran (Fig 5A,B), and
infected with Salmonella WT, a SPI2 strain or mock infected.
Fig. 4. Dynamics of LAMP-1 compartments and SIF in Salmonella-infected cells. HeLa cells were transfected
with LAMP-1-GFP and infected with a S. typhimurium WT strain constitutively expressing GFP. Time lapse
microscopy of GFP fluorescence was performed in intervals of 3 min. starting 1 h after the infection of the cells.
Image acquisition was done using the Perkin Elmer UltraView RS spinning disc confocal microscope. The still
images show projections of 9 Z sections with a spacing of 0.5 m. The micrographs correspond to the time lapse
movie (Movie 3) and time points of acquisition are indicated as hh:mm:ss after infection. Three individual
bacteria that developed into intracellular microcolonies during the course of the experiment are indicated by
arrows (red, orange, yellow). The appearance of tubular vesicular structures positive for LAMP-1-GFP is
indicated by arrowheads of different color. Blue arrowheads indicate SIF that cannot be associated with one of
the three microcolonies. Note the growth, shrinkage and disappearance of SIF starting at 03:30:00 and the
formation of an extensive network of SIF starting at 06:50:00. Division of the intracellular bacteria was
detectable starting at 02:40:00. The perimeter of one infected cell is indicated by a yellow line in the 01:00:00
image. Scale bars, 5m.
46
We observed that SIF were formed in cells infected with the WT strain, but not after
infection with the SPI2-deficient strain or mock infection (Fig 5A). The SIF showed rapid
growth, branching or contraction similar to the phenotype observed in HeLa cells. In non-
activated macrophages, no SIF formation was detectable and we consider the more spherical
cell morphology as main obstacle in visualization of SIF that may also be formed in non-
activated macrophages. Tubular endosomal compartments are commonly observed in living
eukaryotic cells. Although short tubular compartments were found on SPI2 or mock infected
macrophages, the tubular compartments in WT infected cells were clearly distinguishable
with respect to their length and dynamic properties (Fig 5A, Movie S4).
The quantification of the length of fluid tracer-labeled tubular endosomes demonstrates
the unique properties of tubular endosomes induced by WT Salmonella (Fig 5B). The
formation of SIF and dynamic properties of SIF were similar in primary peritoneal
macrophages from mice (Fig. S 5C) and in cell line macrophages (Fig. S 5B).
In contrast to epithelial cells, the formation of SIF in activated macrophages started at
later time points, and was detected after 6 h of infection. Despite the delayed onset, SIF in
macrophages were similar in their dynamic properties to SIF in HeLa cells. Rapid growth,
contraction, branching and fusion of SIF was detected. We also infected RAW cells with a
SPI1-deficient strain (data not shown). The bacteria were either grown to late log phase as for
invasion of epithelial cells, or to stationary phase as used for infection of macrophages with
WT Salmonella. The growth phase of the bacteria had no effect on the time point of onset of
SIF formation, also with exponentially growing bacteria earliest SIF formation was observed
6 h after infection. The number, appearance and dynamics of SIF were indistinguishable in
cells infected with Salmonella WT or the SPI1-T3SS-deficient strain. After invasion of non-
phagocytic cells by Salmonella, the SPI1-T3SS remains active in translocation of effector
proteins from Salmonella within the SCV. A recent study proposed that activity of SPI1
effectors contribute to the biogenesis of the SCV and might act synergistically with SPI2-
T3SS effectors (22). Our observations suggest that, at least in macrophages, there is no
requirement of the SPI1-T3SS and its effectors for the formation of dynamic SIF.
47
Dendritic cells are phagocytic and highly efficient antigen-presenting cells (23). We
previously described that Salmonella is internalized by murine bone marrow-derived dendritic
cells (BM-DC), where the bacteria persist as a non-replicating population (24). However,
Salmonella actively translocates effector proteins by the SPI2-T3SS and affects antigen-
presentation by BM-DC (25). Here we used BM-DC in an experimental setup with fluid phase
tracers and observed extensive tubular structures in Salmonella WT-infected BM-DC (Fig 5C).
SIF rapidly extend, move and contract
We next performed time lapse microscopy of infected HeLa cells with a higher
temporal resolution (1 to 8 images per sec.). The investigation of individual SIF showed the
highly dynamic properties of SIF as shown in Fig 6 (A-D) and Movie 5. Within few seconds
the appearance of SIF tips changes dramatically. Linear growth of SIF was observed as well as
apparently random changes in the direction of SIF extension (Fig 6A).
48
Fig. 5. Salmonella induces highly dynamic SIF in various host cells types. Infection experiments were performed
with the murine macrophage-like cell line RAW264.7 after activation by 5 ng/ml IFN (A, B), or murine bone
marrow-derived dendritic cells (BM-DC) (C). Cells were mock infected or infected with Salmonella WT or SPI2
strains expressing GFP (green). IFN-activated RAW264.7 cells (A, Suppl. Movie 4) were used for infection
with Salmonella WT and the fluid phase marker Alexa568-dextran was used to follow SIF formation in living
cells using a PerkinElmer spinning disk system. A) Left panels show the overview of the infected cells (scale
bars, 5 m) and boxes indicate positions in the periphery of the cells where representative stills show the details
of dynamic alternations of SIF (scale bars, 1 m). The relative time is expressed as hh:mm:ss and SIF growth is
indicated by yellow arrowheads. B) IFN-activated RAW cells were infected as in A) and the length of fluid
tracer-labeled tubular compartments was determined. The average tubule length (±SD) of about 50 to 75 cell per
condition is shown. C) For BM-DC, the fluid phase marker 10 nm gold-BSA-rhodamine was added 1 h prior
infection. Living cells were imaged 8 h after infection using a Zeiss Axiovert 200M wide field microscope. Scale
bars, 5 m.
On individual SIF, phases of extension were directly followed by the complete contraction of
the tubular extension (Fig 6B). Some short tubular vesicles pinched off from longer SIF,
moved in various directions in the cell and could also contact other SIF (see Fig 6C).
SIF were not always connected to SCV. Projections of Z stacks were generated and
indicated that a proportion of the SIF had no detectable contact to any SCV present in the
infected cell. Occasionally, SIF emerged from an SCV, and later lost the connection to the
SCV from which the tubule originated. Also, SIF without initial connection to and SCV were
observed to fuse with SCV (data no shown).
For a subset of SIF, the presence of thinner, LAMP-1-GFP-positive tubular structures
was observed that were followed by a tubular structure with the diameter typical for SIF (Fig
6D, Movie 6). During SIF extension and contraction, the thicker 'trailing' tubules always
followed the thinner 'tracking' tubules. Due to the limitations in resolution, it was not possible
to distinguish if two tubular structures of different diameter were colocalized, or if the thinner
structures temporarily increase or decrease in diameter. This phenomenon was only observed
in cells early after the onset of SIF formations, i.e. 4 - 6 h after infection.
49
SIF extend and contract with different velocities
We first performed time lapse analysis from 1 to 10 h after infection (Fig 4). The
formation of SIF was detectable from 3 h after infection. At later time points, the number of
SIF per infected cell increased, and bacterial replication led to the formation of microcolonies.
The initiation of SIF already 3 h after infection was an unexpected observation, since
previous studies using fixed cells reported SIF formation no earlier than 5 h after infection.
We quantified the appearance of SIF under the various experimental conditions and scored the
appearance of SIF in about 100 infected cells per group. In living LAMP-1-GFP-transfected
cells, 70 % of the cells showed one or more SIF at 4 h after infection and at 8 h after infection,
85 % of the cells showed a complex network of SIF. In fixed cells, about 10 % of the LAMP-
1-GFP transfected cells but none of the non-transfected cells were positive for SIF at 4 h after
infection. Under both conditions, 65 -70 % of cells showed SIF if cells were fixed at 8 h after
infection. The data show that SIF are formed early after infection but these structures are only
visible in living cells and most likely destroyed by fixation.
We set out to quantify the dynamics of SIF at various time points of the intracellular life
of Salmonella. For quantification, various time lapse series were analyzed by recording the
length of individual SIF over time using the EMBL ImageJ plugin Kymograph (26).
Kymographs were generated by determination of the length of selected SIF for each time
point in a time lapse series (see Fig. S 6 for schematic representation and examples).
Extremely dynamic alterations of SIF were observed at early time points after infection
(representative example shown in Fig 7A, Movie7). The kymographs plotted for individual
SIF showed areas of rapid increase of SIF length, followed by rapid contraction, and repeated
extension. Later in infection, i.e. 8 to 10 h after infection, a complex network of SIF extended
through the entire cell. At this time point, the speed of SIF growth and contraction was
reduced (example shown in Fig 7B, Movie 8). The corresponding kymographs were often
'flat', indicating that only minor changes in SIF length occurred over the time of examination.
Kymographs and velocity calculations were performed for a larger number of SIF in cells
infected for 4 to 5 h or cells infected for 8 to 9 h. The velocity of SIF growth and contraction
was calculated and is displayed in Fig 7C. The average speed of SIF growth was 0.4 m/sec.
in cells at 4 to 5 h post infection and reduced to 0.02 m/sec. in cells at 8 to 9 h post infection.
50
As a control of our experimental system, we followed the movement of lysosomes labeled
with lysotracer in living cells at various time points. An average velocity of 1.52 ±m/sec.
and 1.01± 0.85 m/sec. for lysosomes was recorded at 4 h and 8 h after mock infection,
respectively. These data demonstrate that the about tenfold reduction in SIF dynamics was not
due to a general reduction in the velocity of vesicle motility over the time course of the
experiment. We conclude that the dynamics of SIF is inversely correlated with the number of
SIF and the extent of the intracellular replication of Salmonella.
Role of microtubules in SIF dynamics
Previous studies highlighted the role of microtubules (MT) for the biogenesis of SIF (7-
9). The formation of SIF is dependent on the integrity of the microtubule network, as MT
depolymerization by nocodazole prevented SIF formation. The interaction of MT with SIF
was also addressed by ultrastructural analyses (Fig 8A). Here we show that a subset of SIF
forms along microtubules which may act as guidance for the growth of SIF. In certain
instances, SIF were closely associated with two MT. Also, the average velocity of SIF
contraction was substantially reduced from 0.5 �m/sec. to 0.08 �m/sec. at 4 to 5 h and 8-9 h
after infection, respectively (Fig C).
We analyzed the effect of pharmacological inhibitors of MT on SIF dynamics in live
cells (Fig 8B, Movie 9). The addition of the MT-depolymerizing drug nocodazole resulted in
the disintegration of the MT cytoskeleton (Fig. S 7). Nocodazole treatment at 5 h after
infection did result in the loss of a large number of existing SIF (for example, SIF 02 in Fig
8C). However, about 10 to 20 % of the SIF formed prior to nocodazole addition were
maintained after drug treatment (for example, SIF 01 in Fig 8C).
In contrast, most SIF were maintained if nocodazole was added 8 h after infection
(data not shown), i.e. at a time point when the dynamics of SIF were already reduce. SIF were
entirely static presence of nocodazole, while SIF in mock-treated control cells were highly
dynamic at this time point. Similar results were obtained with taxol, an inhibitor that results in
stabilization of MT. To quantify the effects of nocodazole, we performed analyses on SIF in
individual cells prior and after addition of the drug (representative example shown in Fig C).
A kymograph obtained for a SIF prior to nocodazole addition indicated phases of rapid
growth and contraction. After addition of nocodazole, the dynamic changes of the specific SIF
were completely blocked as early as 20 min. after the addition of the inhibitor and the SIF
maintained their length without significant changes for the rest of the observation period.
51
Fig. 6. Dynamic properties of SIF formation. A) to D) HeLa were transfected with LAMP-1-GFP and SIF
formation was followed in living cells 5 h after infection with Salmonella WT expressing GFP using a Zeiss
Axiovert 200M wide field microscope (A, B) or the PerkinElmer spinning disk confocal system (C, D). Details
of representative SIF phenotypes are shown and the relative time points are expressed as hh:mm:ss. Scale bars, 1
�m. A) Extension and branching of SIF was followed. Orange and yellow arrowheads indicate the tips of two
individual SIF. The blue arrowhead indicates a new branch of a SIF. Note the contraction of the SIF labeled by
the yellow arrowhead and the multiple events of branching of the SIF labeled by the orange arrowhead
(corresponding to Suppl. Movie 5). B) The contraction of a SIF was followed. The arrowhead indicates the
retracting tip of a SIF (Suppl. Movie 5). C) Movement of a short SIF. Yellow and orange arrowheads indicated
the leading and trailing end, respectively, of a tubule that pinched off a SIF (not shown), moves and is finally
associated with a longer SIF. D) Dynamic variations of the diameter of SIF. Various examples of SIF that show
variations in the diameter of the tip and central portion of SIF. In the upper panel, the two tips of a branched SIF
are indicated by yellow and orange arrowheads. In the middle and lower panel, note that a tubular filament of
larger diameter (orange arrowheads) follows a tubular filament with smaller diameter indicated by white
arrowheads (Suppl. Movie 6). Scale bar, 5m
52
Fig. 7. Dynamics of SIF at various time points after infection. Transfection and infection was performed as
described for Fig. 4 and time lapse microscopy on the Ultra View spinning disk system was performed with
intervals of 250 ms at various time points after infection. Representative cells at 4 h or 8 h after infection are
shown for the different time points of observation. For the quantification of the velocity of SIF extension,
individual SIF were selected and analyzed using the EMBL ImageJ plugin Kymograph. A) The left panel shows
an infected cell 4 h after infection. The region in the periphery of the cell with multiple SIF is indicated by the
box and shown in detail in the right panel (also shown in Movie 7). The kymograph (lower left panel)
corresponds to SIF #5 and includes the analyses of a 928 frames acquired over a period of 100 sec. Detail
micrographs (lower right panels) show the morphology of SIF #5 during phases of extension (green arrows) and
contraction (red arrows) within the observation period of 100 sec. B) A representative cell is shown 8 h after
infection (also shown in Movie 8). The positions of SIF #1 to #4 are indicated in the upper right micrograph. The
kymographs corresponding to SIF #1 to #4 are shown in the lower panels and have been generated by analyses
of 928 frames acquired over a period of 100 sec. C) The velocity of SIF extension and collapse was calculated
from kymographs generated for cells after 4-5 h or 8-9 h after infection and the data are displayed as box and
whisker plot. Velocities were determined for 50 to 70 events per category. Scale bar, 5m
Finally, we analyzed the effect of the removal of nocodazole on SIF dynamics (Fig D,
Movie 10). As expected, SIF dynamics ceased after addition of the drug, but rapid extension
and retraction of SIF was restored by washing out of nocodazole. At early time points after
nocodazole removal (Fig D, 10 min) we often found dynamic SIF with increased diameter,
but a normal SIF appearance was restored at later time point.
53
Discussion
The intracellular activities of Salmonella enterica result in the remodeling of the host
cell endosomal system and SIF formation. SIF are unique morphological alterations of
endosomes that are only observed in Salmonella-infected cells (7) and dependent on the
function of the SPI2-T3SS (10) and a subset of its effector proteins. Here we describe for the
first time the biogenesis of SIF in living Salmonella-infected host cells and their highly
dynamic properties.
Formation of tubular organelles is a common phenomenon in mammalian cells and
observed, for example, for transport compartments from Golgi to plasma membrane (27) or
sorting endosomes (28). Recently, the formation of tubular vesicular structures with a high
content of major histocompatibility complex (MHC) II was observed in DC (29). Such
tubules are formed without the involvement of intracellular bacteria.
During our live cell analyses, we also observed shorter, motile LAMP-1-GFP-positive
or fluid tracer-labeled tubules in HeLa cells, or RAW macrophages and BM-DC, respectively.
The comparison of cells infected with WT and SPI2 Salmonella or mock-infected cells
revealed the morphological difference between SIF induced by Salmonella and the intrinsic
tubular comportments. The tubular endosomal structures observed in Salmonella WT-infected
BM-DC had dynamic properties and due to their length were clearly distinguishable from
shorter tubular endosomes that appeared in non-infected BM-DC or cells infected with a
SPI2-deficient strain (Fig 5B).
Our observations indicate that dynamic properties of SIF are a general phenomenon
associated with the intracellular life cycle of Salmonella in various host cell types. Uptake of
Salmonella by macrophages is independent from bacterial invasion and function of effectors
translocated by the SPI1-T3SS. Since SIF induction and dynamic properties of SIF were
observed after uptake of non-invasive WT bacteria as well as for SPI1-T3SS deficient strains
but not with SPI2-T3SS-deficient Salmonella, we propose that the phenotypes described here
are independent of SPI1 function.
54
Fig. 8. SIF dynamics is dependent on microtubules dynamics. A) Ultrastructural analyses of SIF in association
with microtubules. HeLa cells were infected and processed for transmission EM as described for Fig. 3. A
representative section with a co-alignment of SIF with microtubules is shown. Arrowheads indicate the close
association of SIF with microtubules (MT). Scale bars 1m (left panel) and 200 nm (right panel). B) HeLa cells
were transfected with LAMP-1-GFP and infected with Salmonella WT expressing GFP. Between 5 to 6 h after
infection, 5g/ml nocodazole, 5 g/ml taxol or an equal volume of the solvent DMSO (mock) were added as
indicated. Stills corresponding to Suppl. Movie 9 are shown that indicate the effect of the drugs on SIF
dynamics. Images were acquired with the Axiovert wide field microscope. Scale bar, 5m. C) The dynamics
properties of selected SIF were recorded in a single cell before and after addition of nocodazole. The left panel
55
shows the infected cell with SIF formation and the section analyzed is indicated by a box. Kymographs were
plotted for SIF #01 prior to addition of nocodazole and at 20, 30 or 40 min after addition of the drug. The fate of
one SIF was followed and analyzed by generation of kymograph prior addition of the inhibitor or 20, 30 or 40
min after addition of the nocodazole. Time lapse series were recorded at 4 frames/sec. for 60 sec using the Ultra
View spinning disk system. Kymographs show merged stacks of 230 frames. D) Removal of nocodazole restores
SIF dynamics. At 5 h after infection with Salmonella WT, HeLa cells were treated with 5 g/ml nocodazole for
30 min. Subsequently, cells were washed twice with PBS, imaging medium was added and incubation was
continued. Time lapse series were recorded of addition of the drug and 10, 20 or 30 min after the wash.
Representative micrographs are shown that correspond to Suppl. Movie 10. Scale bar, 5m
We found that highly dynamic SIF are formed in activated macrophages as well as in
DC. While activated macrophages restrict the replication of intracellular Salmonella, the
bacteria persist as a static, non-replicating population in DC (24). These data clearly
demonstrate that SIF formation is not per se linked to the rapid intracellular replication of
Salmonella as previously proposed (20). A low number of bacteria in DC were sufficient to
induce formation of dynamic SIF without a requirement for intracellular replication. Dramatic
differences in the velocity of SIF extension and contraction at different time points after
infection were recorded. Early after the onset of SIF formation (3.5 - 5 h after infection), the
velocity and the variability in SIF appearance was most prominent, while at later points (> 7 h
after infection), a complex network of SIF was established that exhibited only little extension
or contraction. The simplest explanation is the continuous integration of the endosomal
membranes into SIF, resulting in a shortage of available membranes at later time points of
infection. We observed an inverse correlation between the number of globular LAMP-1-
positive vesicles and the complexity of the SIF network ( Fig. 2). However, one should be
aware that due to the transfection approach applied; only a subset of the endosomal vesicles in
the host cells could be visualized. In addition, host cell molecules mediating the fusion of
vesicle to extending SIF might be titrated by the increasing number of bacterial effector that
are translocated over time. We have previously reported that intracellular Salmonella can
induce, in a SPI2-T3SS dependent manner, the bundling of microtubules of infected host cells
(9). Our ultrastructural analyses showed that SIF can form along MT and that SIF can be
attached to two or more microtubules (Fig 8A).The cross-linking activity might be mediated
by effector proteins that are present in the membranes of SIF and bind directly to
microtubules or indirectly via microtubule-associated proteins.
SIF are considered as the result of continuous aggregation of endosomal membrane
vesicles into tubules of uniform thickness. Our observation of the rapid extension and
56
contraction or collapse of SIF during the early phase of infection would also support other
models (see Fig 9). During the early phase of intracellular life of Salmonella, SIF grow out
from the SCV by both continual fusion of vesicles with the tip of SIF and by pulling force
generated by MT motors associated with the tips of SIF. SIF growth can be directed towards –
ends or +ends, depending on the proportion of dynein or kinesin motors, respectively, that are
recruited. Here, every fusion step will introduce membrane material into the SIF and partially
and temporarily relax the internal stress. However, when the membrane tip of SIF is pulled by
MT motors too far without fusion, the elastic stress in the membrane increases and reaches a
critical threshold.
Fig. 9. Models for dynamic extension and collapse of SIF. A) Salmonella within the SCV translocate effector
proteins (E) of the SPI2-T3SS. B) By activity of effector proteins, membrane vesicles transported on MT are
recruited to, and fuse with, the SCV. These events allow the enlargement of the SCV, delivery of luminal content
to the SCV (indicated by blue shading) but also lead to accumulation of motor proteins on the SCV. C) Increased
accumulation of motor proteins results in a pulling force on the SCV membrane and formation of tubular
extension. D) If pulling forces are too high, motor proteins could loose contact to SIF membrane or MT tracks,
resulting in collapse of SIF. Dependent on the nature of the motor protein recruited, SIF extend towards the – or
+ end of MT. The extension towards the + end appears to be dominant.
57
If motors detach from MT or from SIF membrane, the SIF collapses rapidly relaxing
membrane elastic stress. The higher velocity of SIF contraction compared to extension would
be in line with such model. Although SIF extension in both direction can be observed,
extension towards the +ends dominates over the time course of infection indicating a
preferential recruitment of kinesin or cargo transported by kinesin. The appearance of a
stabilized SIF network in later phase might indicate a consumption of vesicles available for
fusion. Furthermore, the growth is limited by the length of the MT transport track. In addition,
the accumulation of SPI2 effector proteins leads to aggregation of MT that might limit the
transport and stabilize a network of SIF. There is a clear role of MT-based motility, as
indicated by the effect of nocodazole or taxol on SIF dynamics and the function of dynein and
kinesin motors has been reported (15, 16, 30). The specific contribution of individual motor
proteins in the dynamics of SIF extension and contraction remains to be clarified by future
live cell studies. Also, our model cannot explain why SIF without connection to the SCV
appear and move in infected cells.
Depolymerization of MT by nocodazole had different effects on SIF at early and late
time points after infection. In general, SIF dynamics was ablated by nocodazole inhibition.
However, the less dynamic networks of SIF 8 h after infection and later were maintained,
while most of the highly dynamic SIF in the early phase were lost after nocodazole treatment.
This observation might indicate structural differences between 'early' dynamic SIF and 'late'
non-dynamic SIF that we will investigate in subsequent studies.
Role of SPI2-T3SS effector proteins in controlling the MT motor protein activities
acting on the SCV has been reported (31, 32). According to current models for the function of
SPI2-T3SS effector proteins, the SCV has to maintain a balance between opposing activities
of motor proteins (17, 18). Such balance would allow the proper intracellular positioning of
the SCV and the sufficient supply of endosomal membranes to maintain the SCV with an
increasing bacterial population.
These models might have to be reconsidered in the light of our observations of the
highly dynamic nature of SIF. For example, the proposed function of SifA as an effector that
interacts with SKIP in order to prevent the kinesin motor protein activity on the SCV (31)
might not be sufficient to explain the induction of SCV tubulation and the rapid extension or
contraction of SIF.
58
Future studies have to reveal the contribution of MT motor proteins to the dynamic
features of SIF and how these activities are manipulated by the function of a subset of SPI2-
T3SS effector proteins. The function of the SPI2-T3SS has been linked to various intracellular
events such as the avoidance of antimicrobial activities, modification of the MT and actin
cytoskeleton, interference with antigen presentation and many others (reviewed in 5, 6).
The biological role of the induction of SIF and the remodeling of the endosomal
compartment of the host cell of Salmonella is not completely understood. A clear requirement
for intracellular replication is the recruitment of membranes for the extension of the SCV
(10). We applied various methods for the labeling of the luminal content of SIF as well as the
membranes and both approaches resulted in the appearance of SIF with similar morphology
and dynamic properties. Interestingly, we could observe that the lumen of SIF as well as of
the SCV was accessible to fluid phase markers in various Salmonella-infected host cells. A
recent study by Drecktrah et al. (19) used similar labels and a live cell setup and these authors
observed the continuous interaction of the SCV with endocytosed material. The data reported
here support the model of Drecktrah et al (19) and stand against the currently prevailing
model that the SCV is separated from the endosomal system. We propose that Salmonella
within the SCV experiences nutritional limitations but temporarily gains access to external
material by induction of SIF. Future work has to reveal whether this also leads to an increased
availability of nutrients that may otherwise be limited within the SCV. We will also
investigate if this recruitment contributes to the nutritional status of Salmonella in the SCV
and could explain the unique intracellular lifestyle of the pathogen.
Materials and Methods
Bacterial strains and culture conditions
Salmonella enterica serovar Typhimurium (S. typhimurium) NCTC 12023 was used as wild–
type strain. Experiments were performed in part with S. typhimurium LT2A, a strain which
shows attenuated virulence in vivo. We found no difference in the intracellular survival and
replication or the SIF induction between 12023 and LT2A (Fig. S 1). For live imaging, strains
were used harboring plasmid pFPV25.1 (33) or pFPV-mCherry/2 (kindly provided by L.A.
Knodler) for the constitutive expression of eGFP or mCherry, respectively.
Mutant strains invC, P2D6 and P3H6 defective in the SPI1-T3SS, SPI2-T3SS or effector gene
sifA, respectively, have been described before (10, 34). Bacterial strains were routinely
cultured in LB with addition of 50g/ml carbenicillin of required to maintain plasmids.
59
Cell culture
Human epithelial cell line HeLa cells (ATCC No. CCL-2) and the murine macrophage-like
cell line RAW 264.7 (ATCC No. TIB-71) were cultured in DMEM with 10 % FCS, penicillin
and streptomycin, and grown in 37°C with 5 % CO2. Cells were cultured in medium without
antibiotics prior to bacterial infection. For the activation of RAW264.7 cells, interferon �
(IFN�) was added in concentrations between 2 to 10 ng/ml for 24 h. Typically, 5 ng IFN�
/ml were used and resulted in about 90 % activated macrophages as judged from the cell
morphology. For the generation of primary peritoneal macrophages, BALB/c received and
intraperitoneal injection of 4 % thioglycolate in PBS and after 4 d, macrophages were isolated
basically as described before (35). Murine bone marrow-derived dendritic cells (BM-DC)
were generated from C57BL/6 mice essentially as described before (24). BM-DC was further
enriched by sorting on MACS columns (Milteny Biotech) and a purity of 90 % was routinely
obtained. The BM-DC were in an immature state (CD11c high, MHC II low) as confirmed by
FACS analyses.
Host cells infection
For infection of HeLa cells, bacterial strains were grown overnight in LB broth. Overnight
cultures were diluted 1:30 in fresh LB broth and sub-cultured for 3.5 h. At this time point the
cultures reached the late log phase and were invasive.
HeLa cells were infected an MOI of 50. For infection of RAW264.7 and murine peritoneal
macrophages, as well as for BM-DC, the strains were grown for 14-16 h. Such overnight
cultures were diluted and used directly for infection of macrophages or BM-DC at an MOI of
10 or 50, respectively.
After an incubation of 30 min to allow bacterial invasion, the cells were washed thrice with
PBS to removed non-internalized bacteria. Subsequently, DMEM containing 10 % FCS and
100 �g gentamicin/ml was added to kill non-internalized bacteria. After incubation for 1 h,
the medium was replaced by medium containing 10 �g/ml gentamicin for the rest of the
incubation time.
Transfection
The plasmid for the eukaryotic expression of LAMP-1-eGFP was kindly provided by Patrice
Boquet. HeLa Cells (2 x 104) cells were seeded in an 8 chamber glass slide (Nunc-LabTek)
and allowed to adhere overnight. Transfection was routinely performed by the calcium
60
phosphate methods (36). Here, 500ng of plasmid DNA (LAMP-1 for single transfections)
were mixed with the transfection reagent and added to cells in 8 chamber slide with DMEM
with 10 % FCS. Cells were incubated for 4 to 5 h and later medium was changed and fresh
DMEM with 10 % FCS was added, cells were later used for infections 16 to 18 h after
transfection.
Live cell imaging
Cells were cultured in chamber slides and transfection or stimulated with IFN� if
required. Infection with Salmonella strains was performed as described above and
extracellular bacteria were killed by incubation with medium containing 100�g/ml
gentamicin. After 1 h, this medium was replaced by imaging medium containing 10�g/ml
gentamicin. Imaging medium is Eagles Minimum Essential Medium (MEM) without L-
glutamine, phenol red and sodium bicarbonate, containing 30 mM HEPES, pH 7.4. The
chamber slide was then taken for imaging at required time points after infection. The chamber
slide was mounted on the microscope stage equipped with a humidified environment chamber
maintaining 37°C and 5 % CO2. For inhibitor studies, nocodazole (Sigma) or placitaxel
(Calbiochem) were used at the indicated concentrations from stock solutions in DMSO. The
effect on the inhibitors on MT was controlled by immunofluorescence (Fig. S 5).
Fluid phase marker and pulse chase
For tracing the endocytic pathway, various fluid phase markers were used. Alexa Fluor
568- conjugated dextran 10,000 MW, anionic, fixable (Molecular probes D-22912) was
obtained from Molecular Probes, Invitrogen. A 10 nm gold BSA conjugate was generated as
previously described (37). Subsequently, gold BSA was fluorescently labeled with NHS
rhodamine succinimidyl ester (Molecular Probes, Invitrogen). For use as tracer in live cell
experiments, the solution was adjusted to an OD520 of 0.1 and aliquots of 100�l were added
to the wells of chamber slides at the indicated time points. For some experiments, HeLa cells
were transfected prior infection with a LAMP-1-GFP vector, otherwise non-transfected cells
were used. HeLa cells were infected by invasive Salmonella strains as described and 4 h post
infection, cells were incubated with 200μg/ml Alexa568-dextran or gold-rhodamine for 3 h
and subsequently washed. Cells were incubated for the rest of the experiment with label-free
media and subjected to live imaging. Macrophages were infected and 6 to 7 h post infection,
cells were incubated with 100μg/ml Alexa568-dextran for 20 min., cells were washed thrice
with PBS and then incubated with DMEM with 10 % FCS without tracer for 30 - 40 min.
Subsequently, the cells were used for live imaging. For BM-DC, the tracers were added prior
61
infection, incubated for 20 min, followed by washing of the cells and infection with
Salmonella. For labeling of lysosomes, HeLa cells were then washed once with PBS and
loaded with 500nM Lysotracker Red DND-99 (Molecular probes, L-7528) in DMEM with
FCS for 1 h. After pulsing, the cells were washed thrice with PBS and 500 �l of imaging
medium was added to each well and the cells were imaged.
Microscopy and imaging
Imaging studies were done using two spinning disc confocal microscopes (UltraView
RS and ERS, PerkinElmer). The spinning disk head (Yokogawa) of both systems was
mounted to an inverted microscope (Axiovert200, Zeiss) which was equipped with a 100 x
Plan Neofluar objective (Zeiss). For 4D image acquisitions, a microscope was used with an
acoustic optical tunable filter for wavelength selection (AOTF), a highly sensitive CCD
camera, and 5 laser lines along with a Nipkow spinning disc for high temporal acquisition
with stacks or 4D imaging. The microscope was kept at 37°C by a microscope incubator box
(EMBL Heidelberg).
At various time points post infection images were taken of cells infected with
Salmonella. GFP fusion proteins were excited with the 488 nm line of an argon ion laser.
Alexa568-detran and mCherry fusion proteins were imaged using either the 568 nm line of a
krypton gas laser (UltraviewRS) or a 560 nm diode laser (UltraviewERS). The exposure time
and the Z-spacing were adjusted individually for each cell/condition and are given within the
legend of the Figs. Additionally some of the time series were also recorded using an inverted
fluorescent microscope (Zeiss Axiovert 200M, equipped with Axiocam MRm (Zeiss), and 100
x Plan Neofluar oil immersion objective (Zeiss). The system was equipped with an incubation
chamber and heating unit (Zeiss) which maintained 37°C, 5 % CO2 and humidity during live
imaging. The microscope has a mercury lamp 100W (Zeiss) as fluorescence light source and a
motorized filter set for various flurochromes (GFP, Cy5, DAPI, FITC etc).
All acquisitions and settings for the live imaging were using the Axiovision 4.5 software
(Zeiss, with various modules). The resulting movie series were corrected for background
fluorescence and bleaching using bleach correction and background subtraction macros
available at EMBL Image J. Majority of the image analysis (to calculate the velocity of SIF
growing and shrinking and velocities of LAMP-1 vesicles was done using the macro
Kymograph written for EMBL Image J (26) (available at
http://www.embl.de/almf/html/downloads.html) and also manual tracking.
62
Electron microscopy and immuno-labeling
For plastic (EPON) sections: HeLa cells were grown on glass cover slips in plastic
culture dishes in complete DMEM medium. Cells were infected with S. typhimurium at MOI
= 50 and incubated for 10 h prior fixation. All samples were fixed 10 h post infection in 2 %
glutaraldehyde in 0.1 M cacodylate buffer (CB), post-fixed in 2 % osmium teroxide,
dehydrated in ethanol series and propylenoxide, flat-embedded and polymerized in EPON812
(Serva). Glass coverslips were removed in liquid nitrogen and blocks with cell monolayer
were embedded and polymerized again to enable cutting in parallel or perpendicular to Z axis.
Sections (40 nm) were cut with diamond knife on Reichert Ultracut S and contrasted in uranyl
acetate and lead citrate.
For cryosections, HeLa cells grown on plastic culture dishes in DMEM complete
medium were transfected (or mock-transfected) with the LAMP-1-GFP vector. During
transient expression, cells were infected (or mock-infected) with S. typhimurium expressing
GFP and incubated for 10 h prior fixation. Samples were fixed with 4 % PFA and 0.05 % GA
in 0.1 M Sorensen phosphate buffer, pH 7.4, scraped gently with a rubber policeman, washed
and embedded in low melting 10 % gelatine (Sigma). Small blocks were cut with razorblade,
infiltrated with 2.3 M sucrose, mounted on aluminum pins and snap-frozen in liquid nitrogen.
Blocks were trimmed at -90°C and cut at -120°C with 90° diamond knife (Diatome,
Switzerland) using Leica FC6 cutting machine. Sections were collected in drop of sucrose-
methylcellulose and transferred on carbon-coated CuPd EM grids with parlodion film. For
immuno-labeling, we used mouse anti-LAMP-1 (clone H4B4, DSHB, Iowa City) followed by
Goat-anti mouse secondary antibody conjugated to 10 nm gold (Biocell Int.) or with rabbit
anti-GFP (Molecular Probes) followed by Protein A–coupled with 15 nm gold (CMC
Utrecht). Grids were contrasted and embedded in mixture of Uranyl Acetate-methylcellulose.
Samples of plastic sections or immuno-labeled cryosections were observed with a Phillips
Morgagni EM 268D operating at 100 kV. Pictures were taken using CCD camera and
AnalySIS 3.2 software. Final Figs were edited with Adobe Photoshop 8.0.
Acknowledgements
This work was supported by grants HE1964/9-2 and 9-3 of the Deutsche
Forschungsgemeinschaft to M.H. We like to thank Rainer Pepperkok, Trina Schroer and Garth
Abrahams for support and stimulating discussions regarding this work and Stefan Terjung and
Yury Belvaev for support. The support of Sebastian F. Zenk and Barbara Bodendorfer for
63
generation of DC and primary macrophages is gratefully acknowledged. The continuous
support of the Advanced Light Microscopy Facility at the EMBL by PerkinElmer is gratefully
acknowledged.
Abbreviation list
DC, dendritic cell; MT, microtubule; SIF, Salmonella-induced filaments; SPI1, Salmonella
Pathogenicity Island 1; SPI2, Salmonella Pathogenicity Island 2; SCV, Salmonella-containing
vacuole, T3SS, type III secretion system WT, wild type.
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cytochemistry. Eur J Cell Biol. 1985;38:87-93.
Supplementary Material
Dynamic Remodeling of the Endosomal System during Formation of Salmonella
Induced Filaments by Intracellular Salmonella enterica
Roopa Rajashekar, David Liebl, Arne Seitz, and Michael Hensel
67
Fig. S 1. Intracellular replication of various Salmonella enterica strains and SIF induction. A) The intracellular
replication of S. typhimurium strains LT2A and 12023 was compared. HeLa cells were infected with wild-type
(WT) or SPI2-deficient strains (ssaV) and non-internalized bacteria were killed by addition of Gentamicin. The
number of viable intracellular bacteria or colony-forming units (CFU) was determined at 2 h and 16 h after
infection by lysis of host cells and plating of lysates onto agar plates. The –fold intracellular replication was
determined by the ratio of CFU counts at 16 h and 2 h after infection. B) LAMP-1-GFP-transfected HeLa cells
were infected with GFP-expressing Salmonella WT 12023 or LT2A as indicated. Live cell imaging was
performed 7 h after infection and representative stills of a time lapse series are shown. Scale bar, 10 m.
Fig. S 2. Localization of LAMP-1-GFP and endogenous LAMP-1 in Salmonella-infected cells. HeLa cells were
transfected with the LAMP-1-GFP construct (green) and infected with Salmonella WT expressing GFP (green). 6
h after infection, the cells were fixed and processed for immuno-staining of LAMP-1 (red). The position of
intracellular Salmonella and SIF is indicated in the merged image by arrows and arrowheads, respectively. Scale
bar, 5 m.
Fig. S 3. Quantification of LAMP-1-positive vesicles. HeLa cells were transfected with the LAMP-1-GFP
construct. A representative still is shown from a time lapse series. The quantification of the number of spherical,
LAMP-1-GFP-positive vesicles was performed manually using the Edit mode of EMBL ImageJ. Scored
compartments were marked by yellow dots and the total number of events is indicated. This approach was used
for the quantification shown in Fig. 2B. Scale bar, 5 m.
68
Fig. S 4. Ultrastructure of the endosomal system in mock-infected cells. HeLa cells were mock infected in
parallel to infection with WT Salmonella and processed as described for Fig. 3C. Representative mock-infected
cells are shown. Scale bars, 1 m.
69
Fig. S 5. Salmonella induces highly dynamic SIF in various host cells types. Infection experiments were
performed with the murine macrophage-like cell line RAW264.7 after activation by 5 ng/ml IFN (A). Cells
were mock infected or infected with Salmonella WT or SPI2 strains expressing GFP (green). The fluid phase
marker 10 nm gold-BSA-rhodamine was added 5 h after infection. Living cells were imaged 8 h after infection
using a Zeiss Axiovert 200M wide field microscopy. Scale bars, 5 m. IFN-activated RAW264.7 cells (B,
Suppl. Movie 11) or primary murine macrophages (C, Suppl. Movie 11) were used for infection with Salmonella
WT and the fluid phase marker Alexa568-dextran was used to follow SIF formation in living cells imaged 8 h
after infection. Left panels show the overview of the infected cells (scale bars, 5 m) and boxes indicate
positions in the periphery of the cells where representative stills show the details of dynamic alternations of SIF
(scale bars, 1 m). The relative time is expressed as hh:mm:ss and SIF growth and SIF contraction is indicated
by yellow and orange arrowheads, respectively.
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Fig. S 6. Analyses of SIF dynamics by kymographs. A) Schematic representation of the analysis of SIF
extension and contraction. Individual SIF were identified in time lapse series and analyzed by the EMBL ImageJ
plug-in Kymograph. Kymograph (also called time-space plot) is a graphical method of displaying and analyzing
moving structures. To create such a plot the trajectory of the moving object has to be found. Once this is defined
the pixel values of this trajectory are copied to a new image. This procedure is repeated for each frame of the
image stack. If the moving structure is resulting from a fluorescently labeled particle it is represented by a bright
line in the kymograph. The slope of this line is proportional to the velocity of the moving particle and also
dependent on the directionality of the particle. Non moving particle can be identified by vertical or horizontal
lines (depending on the plotting method).Thus it is possible with this method to analyze the speed and
directionality of particles
B) Kymographs for individual SIF recorded at 4-5 h or 8-9 h after infection of LAMP-GFP-transfected HeLa
cells with Salmonella WT. These are examples of the kymographs used to calculate the data shown in Fig. 7C.
Fig. S 7. Effect of addition of Nocodazole on the microtubule cytoskeleton. HeLa cells were treated with a final
concentration of 10 g/ml Nocodazole. The cells were fixed at the indicated time points and processed for
immuno-staining of -tubulin. Scale bars, 5 m
Supplementary Movie Legends
Movie 1 corresponding to Fig. 2A. Movement and fusion of a LAMP-1-GFP-positive vesicle
to the SCV. An overview of an infected cell is shown with a LAMP-1-GFP-positive vesicle
docking to an SCV (indicated by a circle). Scale bar, 5 m.
Movie 2. corresponding to Fig. 2B. Appearance of tubular extensions from an SCV. Scale bar,
5 m.
Movie 3 corresponding to Fig. 4. A long time lapse from 1 h to 10 h after infection show the
formation of Salmonella microcolonies, highly dynamic SIF at early time points after
infection and the appearance of a complex SIF network later after infection. Images were
taken with intervals of 3 min. Scale bar, 5 m.
71
Movie 4 corresponding to Fig. 5A. Interferon -stimulated RAW264.7 macrophages were
infected with Salmonella WT or a SPI2 mutant strains expressing GFP or mock-infected. The
cells were pulsed with Alexa568-dextran 5 h after infection and imaged 2 h later. Detail
sections are shown as indicated by the box in the overview still. Scale bar, 1 m.
Movie 5 corresponding to Fig. 6A and Fig. 6B. Growth, branching and collapse of SIF. HeLa
cells were infected with LAMP-1-GFP and infected with Salmonella WT expressing GFP. The
event shown was observed 4 h after infection. Scale bar, 2 m.
Movie 6 corresponding to Fig. 6D. The dynamic variations in SIF diameter were recorded a
setup described for Fig. 6A.
Movie 7 corresponding to Fig. 7A. Dynamics of SIF formation shown for a representative cell
at 4 h after infection.
Movie 8 corresponding to Fig. 7B. Dynamics of SIF formation shown for a representative cell
at 8 h after infection.
Movie 9 corresponding to Fig. 8B. HeLa cells were transfected with the LAMP-1-GFP
construct and infected with GFP-expressing Salmonella WT. At 4 to 5 h after infection, the
solvent (mock) or inhibitors nocodazole or taxol were added and live imaging of infected cells
was performed.
Movie 10 corresponding to Fig. 8D. The effect of nocodazole treatment and removal on the
dynamics of vesicle and SIF movement is shown.
Movie 11 corresponding to Fig. S5 B and C. RAW264.7 cell (B) or primary murine
macrophages were infected with Salmonella WT and the fluid phase marker Alexa568-detran
was used to follow SIF formation in living cells.
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Chapter-2
3.2 Novel functions of SPI2 effector proteins during intracellular pathogenesis of
Salmonella enterica revealed by live cell and Ultrastructural analyses
Roopa Rajashekar1, David Liebl2, Deepak Chikkaballi1 and Michael Hensel1
Mikrobiologisches Institut, Universitätsklinikum Erlangen1, Cell Biology and Biophysics Unit, EMBL Heidelberg2
Address for correspondence:
Michael Hensel
Abteilung Mikrobiologie
Fachbereich Biologie/Chemie
Universität Osnabrück
Barbarastr. 11
49076 Osnabrück
Abstract
Intracellular Salmonella enterica induce a massive remodeling of the endosomal
system in infected host cells. The host dramatic consequence of this interference is the
induction of extensive tubular aggregations of membrane vesicles, referred to as Salmonella-
induced filaments or SIF. In this study, we applied live cell imaging and electron microscopy
to analyse the role of individual SPI2 effectors. Here we report the role of various effector
proteins of the SPI2-T3SS in the SIF dynamics and their phenotypic appearance. In
accordance with previous observations most of the SPI2 effectors had little or no effect on SIF
formation or dynamics. However in contrast to analyses in fixed cells, the SIF induced by
sseF- or sseG-deficient strains were not discontinuous, but rather continuous and appeared
thinner in diameter as seen in living host cells. A very dramatic difference was observed for a
pipB2-deficient strain that induced very bulky, non-dynamic aggregations of membrane
vesicles. We quantified these results and report here the behavior of some of the well known
SPI2 effectors like SseF and PipB2 in more detail.
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Introduction
Salmonella enterica is a facultative intracellular pathogen that modifies eukaryotic host
cells in order to establish a unique parasitophorous vacuole, the Salmonella-containing
vacuole or SCV. The SCV is a membrane-bound compartment that has several features of late
endosomal compartments and allows the survival and replication of S. enterica in a variety of
mammalian host cells types (Haraga et al., 2008). Of central importance for a successful
intracellular lifestyle of Salmonella is the function of the type III secretion system (T3SS)
encoded by Salmonella Pathogenicity Island 2 (SPI2) (Kuhle & Hensel, 2004). Intracellular
Salmonella deploy the SPI2-T3SS to translocate a large number of effector proteins across the
phagosomal membrane. Collectively, these effector proteins enable the intracellular
proliferation of Salmonella. The molecular functions and the interacting proteins of the host
cell are unknown for the majority of the SPI2-T3SS effectors, and not all effectors appear to
be relevant for the intracellular phenotypes and systemic pathogenesis.
A specific characteristic of Salmonella-infected cells is the massive reorganization of
the endosomal system. The formation of extensive tubular aggregations of endosomal
membrane vesicle has been observed and these structures have been termed Salmonella-
induced filaments or SIF (Garcia-del Portillo et al., 1993). SIF are characterized by the
presence of late endosomal/lysosomal membrane proteins such as LAMP-1. The most severe
intracellular phenotype is mediated by SPI2-T3SS effector SifA. Mutant strains lacking SifA
are highly attenuated in systemic virulence and intracellular replication (Stein et al., 1996).
Bacteria deficient in sifA fail to induce SIF and the bacteria lose the SCV membrane during
intracellular replication thereby escaping into the cytoplasm (Beuzon et al., 2000). SseF and
SseG effectors contribute to the intracellular lifestyle, although the defects in intracellular
replication of the corresponding mutant strains are less pronounced compared to sifA or SPI2-
T3SS deficient strains (Hensel et al., 1998; Kuhle & Hensel, 2002). Further effectors with
reported contribution to the formation and maintenance of the SCV are SopD2 and PipB2. A
role for PipB2 in control of the centripetal extension of SIF was observed (Knodler & Steele-
Mortimer, 2005) and PipB2 was identified as a linker for kinesin (Henry et al., 2006). SseJ
has an enzymatic activity and acts as a deacetylase after translocation into host cells (Ohlson
et al., 2005). For SseL, a function as de-ubiquitinylating enzyme has been observed.
In contrast to SifA, SseF and SseG, the contribution of the other effectors is low or not
detectable. In addition, the virulence in murine models of mutant strains lacking individual
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effectors is usually in good correlation with the ability to survive and replicate inside host
cells. There are several observations that indicate the modification of the host cell endocytic
system and transport processes in infected cells.
Interference of intracellular Salmonella with host cell transport
Central role of the SPI2-encoded T3SS, translocation of a complex set of
effector proteins by Salmonella within the SCV
The initial studies on SIF biogenesis were performed by immuno-staining of cells
fixed at various time-points after infection and these analyses led to the model that SIF
emerge from continuous aggregations of endosomes into large tubular compartments. This
model probably has to be revised by recent analyses of the intracellular fate of Salmonella by
life cell imaging. We and others (Rajashekar et al., 2008) found that the formation of SIF is a
highly dynamic process in various types of host cells. SIF show rapid extension, branching
and contraction in the early phase of intracellular life of Salmonella, while at later stages, a
complex network of SIF can be detected that is highly reduced in dynamics. The indication of
SIF in living host cells and their dynamics properties were entirely dependent on the function
of the SPI2-T3SS and the properties of SIF could be clearly distinguished from other tubular
organelles that are found in phagocytic cells. Based on our initial studies we set out to analyze
the role of various effector proteins of the SPI2-T3SS to the induction of SIF in living host
cells, their dynamics and morphology. Our live cell studies as well as ultrastructural analyses
demonstrate the specific contribution of SifA, SseF, SseG and PipB2 to formation of SIF,
while no contribution was observed for the remaining effector proteins.
Results
Systematic analysis of the role of SPI2 effector proteins in dynamics of
Salmonella-induced filaments
We used our newly established live cell setup (Rajashekar et al., 2008) to analyze the
role of the various effector proteins of the SPI2-T3SS for the intracellular fate of Salmonella
within host cells and the modification of the endosomal system of host cells by WT and
various isogenic mutant strains defective in effector proteins of the SPI2-T3SS. Our
preliminary studies indicated that SIF showed very dynamic extension and retraction early
after the onset of SIF formation, i.e. 4-5 h post infection of HeLa cells. Several hours later, i.e.
8 h post infection and later, the velocity of extension and retraction of SIF was reduced but the
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overall number of SIF was increased. For a systematic comparative analysis we used time
points of 4-5 h and 8-9 h post infection and imaged the effects on the endosomal system in
HeLa cells transfected with LAMP-1-GFP.
A collection of mutant strains was generated that either contained a defect in the SPI2-
encoded T3SS or deletions of genes for individual effector proteins of the SPI2-T3SS. Since
SopB, an effector translocated by the SPI1-T3SS, has been reported to interfere with the
organization of host cell endosomes, the phenotype of a sopB strain was also investigated. All
mutant strains were able to invade HeLa cells. We also observed that the various strains
showed rather similar intracellular characteristic during the first phase of intracellular
presence. In general, an association of the bacteria with the late endosomal/lysosomal
membrane marker LAMP-1 was observed. In accordance with our previous studies, the WT
and SPI2 strains were predominantly found to be enclosed by LAMP-1-positive membranes.
Induction of LAMP-1-containing membrane tubules was observed in WT-infected cells but
not in cells harboring the SPI2-deficient strain. The spiC-deficient strain was phenotypically
indistinguishable from the SPI2 strain. In cells infected with the sifA mutant strain, LAMP-1-
positive membranes were found in the vicinity of the bacteria in the early phase of infection
(4-5 h post infection) but at later time points (7-8 h post infection); an association between the
intracellular bacteria and LAMP-1-postive compartments was usually absent. No formation of
membrane tubules was observed for cells infected with the sifA strain.
We found that the majority of mutant strains defective in one effector showed very
similar intracellular characteristics as observed for the WT strain. In detail, live cell analyses
of HeLa cells infected (Fig 1) with mutant strains defective in sifB, sseL, sseK1, sseK2, sseI,
sseL, sspH1, sspH2, slrP, steC, or pipB were performed. For each of these strains, the
intracellular bacteria were contained in LAMP-1-positive compartments. SIF were induced by
these mutant strains. The morphology of the SIF was identical to SIF induced by WT
Salmonella. The SIF induced by the effector mutant strains showed highly dynamic properties
in the early phase of intracellular life, and less dynamic characteristics in the later stage of
infection. Representative stills from time lapse experiments with the various strains are shown
in Fig 1 and a movie with a collection of the time lapse series for cells infected with WT and
the various mutant strains can be found in the Suppl. materials.
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Fig. 1. Systematic analyses of the role of effector proteins of the SPI2-T3SS for the formation and morphology
of Salmonella-induced filaments in living host cells. HeLa cells were transiently transfected with a vector for the
expression of LAMP-1-GFP. A set of isogenic strains of S. enterica with deletions of specific effector proteins
was used to infect transfected cells. The bacteria harbored plasmids for the constitutive expression of GFP or
mCherry. Time lapse series for transfected and infected cells are recorded for the late phases of intracellular life,
i.e. 8 - 9 h post infection. Still images from representative time lapse series are shown. A collection of the
corresponding movies is available as (Suppl. movie 1). Scale bar, 5m
Integrity of the SCV in living Salmonella-infected cells
In addition to the formation of SIF, we used the live cell setup for screening for the
integrity of the SCV. Previous observations indicated that WT-inhabited SCV were
continuously outlined with LAMP-1-GFP. The continuous labeling of this membrane marker
suggested an intact membrane envelope.
77
We also found that fluid tracers added at various time points after infection outline the
SCV in a rather continuous manner. In contrast, immune-staining of the SCV in fixed cells
often result in a discontinuous labeling of proteins in the SCV, indicated that the structures are
not fully accessible to antibodies or that the fixation results in a disruption of the SCV
membrane. Analyses of living cells allowed a clear distinction of the SCV integrity as
compared to the fixed cells (our unpublished observation). We thus come to the conclusion
that live cells assays fair better in terms of analyzing phenotypes in native form over fixation
protocols.
The sseJ and sseJ/sifA strains
A mutant strain deficient in sseJ has been reported to cause increased tubulation of
endosomal membranes and was reported that the a sifA/sseJ double mutation can compensate
the loss of the SCV observed in the sifA strains (Ruiz-Albert et al., 2002). Live cell imaging
of cells infected with the sseJ strain did not reveal detectable differences in the organization
and dynamics of SIF. We investigated the characteristics of a mutant strain deficient in both
deficient in both sifA and sseJ. In contrast to previous reports based on the analyses of fixed
cells, the investigation in living cells did not indicate that a larger number of intracellular
bacteria were able to maintain the SCV. Similar to the phenotype of the sifA strain, we found
that the sifA/sseJ strain did not induce SIF and lost the association with LAMP-1-containing
membranes in the later phase of intracellular life.
In addition to the sifA mutant, our systematic screen of SPI2 effector phenotypes in
living cells revealed that mutant strains deficient in sseF, sseG or pipB2 were altered in the
effects on the endosomal system of the host cell. The mutant strains will be described in detail
below.
The sseF strain
In cells infected with the sseF strain, and similarly the sseG strain, an aberrant SIF
phenotype has been described (Kuhle & Hensel et al., 2002). The discontinuous structures
observed in fixed cells were referred to as pseudo-SIF to distinguish from the aggregates
induced by WT Salmonella. Pseudo-SIF that appeared as a linear array of spherical vesicles
with a discontinuous staining for LAMP-1 and other endosomal membrane markers (Fig 2B
Lower panel). Using the live cell setup, we observed a different appearance of SIF in cells
infected with the sseF strain. The formation of long tubular aggregates (Fig 2B upper panel)
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(Suppl. movie 3) was observed that were less intensely labeled and appeared to be of lower
diameter compared to the SIF induced by WT Salmonella (Fig 2A) (Suppl. movie 2).
Fig. 2. Intracellular fates of sseF and sseG strains in living host cells. A) And B) HeLa cells were transfected
with the LAMP-1-GFP construct and subsequently infected with S. enterica WT and strains deficient in sseF or
sseG, respectively. B (Lower panel) HeLa cells were infected with sseF GFP mutant and processed for
immunostaining using anti-LAMP-1. C) HeLa cells were transfected with the LAMP-1-GFP construct and
subsequently infected with pipB2 GFP mutant. C) (Lower panel) HeLa cells were infected with the pipB2 mutant
strain expressing GFP and processed for immunostaining using anti-LAMP-1. D) Alternatively, HeLa cells was
infected with WT, sseF or pipB2 strains, respectively and post 3h infection cells were pulsed with Alexa568-
dextran and post 8 h cells were washed and live imaging was performed. E) Mutant strains were complemented
by a plasmid expressing sseF or pipB2. Time lapse series were generated at 8 h post infection. Scale bar, 5m
The overall number of cells that showed tubular aggregation after infection with sseF
Salmonella was lower and the number of tubular structures in the SIF-positive cells was also
79
reduced. The induction of SIF in cells infected with the sseF mutant strain initiated 8 h post
infection, compared to cells infected with Salmonella WT where SIF are induced as early as
3.5 h post infection (Rajashekar et al., 2008). Live cell analyses of infected cells indicated that
the SIF were also dynamic and the velocity of extension and contraction was in a range
similar to that of SIF induced by WT Salmonella.
The pipB2 strain
The most aberrant intracellular phenotype was observed for the pipB2 strain. A subset
of the pipB2 infected host cells showed SIF that were indistinguishable for SIF (data not
shown). However, in other host cells infected with the same strain we observed extremely
large LAMP-1-positive membrane compartments that will be referred to as ‘bulky SIF’ (Fig
2C upper panel) (Suppl. movie 4). Bulky SIF were generally connected to an SCV. In contrast
to SIF with normal appearance induced by the pipB2 strain, bulky SIF were almost static and
the extension or contraction of the compartments was extremely low. In order to further
investigate this phenotype, HeLa cells were infected with pipB2-deficient bacteria without
transfection, and 12 h post infection, cells were processed for immunostaining of endogenous
LAMP-1. Under these conditions, the same bulky aggregates of LAMP-1 positive vesicles
were observed (Fig 2C, lower panel). The onset of SIF in pipB2-infected cells was observed
between 4 to 5 h post infection, similar to the situation in WT-infected cells. The subset of SIF
which appeared on par to WT SIF followed the normal progression of dynamics that is
initially highly dynamic SIF were seen followed by non dynamic network SIF.
SCVs and SIF compartments in sseF- and pipB2-infected cells are
accessible for fluid tracers
It has been recently established that SCV compartment is accessible to the incoming
endosomes by detecting the fluid tracers in these compartments (Drecktrah et al., 2007;
Rajashekar et al., 2008). Pulse chase experiments using fluid tracers, gave an insight into the
interaction of endosomal vesicles with the SCV and SIF. In order to see if there is any
difference in the accessibility of fluid tracers to SCV when infected with sseF and pipB2
mutants, we performed pulse chase experiment using Alexa568-dextran. HeLa cells infected
with WT, sseF or pipB2 strains were pulsed with Alexa568-dextran. As shown in (Fig 2D 1, 2
and 3 panels), there was no significant difference in acquiring Alexa568-dextran by these
mutants as compared to that of WT. Therefore we can conclude that absence of SseF or PipB2
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effector proteins do not hamper the acquisition of endosomes to the SCV and SIF
compartments.
Complementation of sseF and ppB2 mutant strains restores WT phenotype
Both the sseF and pipB2 phenotypes were complemented by a functional copy of the
mutated gene and it could be observed that the classical SIF phenotype was restored, as
shown in (Fig 2E). (Suppl. movie 5)
Dissecting the Pseudo-SIF phenotype
We hypothesized that the different form the visualization of SIF (LAMP-1 staining vs.
LAMP-1-GFP detection) or the analysis of fixed vs. living cells might be the reason for
difference in the appearance of SIF. To test these hypotheses, we first performed the tracing of
SIF in HeLa cells infected with both WT and sseF GFP mutant strain and post 16 h the cells
were fixed with PFA and processed for immunofluoroscence with anti LAMP-1 antibody. As
previously observed a punctuated staining of LAMP-1 was observed in the cells infected with
sseF mutant (Fig 3A see inset) as compared to smooth appearance of SIF in cells infected
with WT (Fig 3B).
In the next step, HeLa cells were transfected with LAMP-1-GFP and infected with
sseF mutant and live imaging was performed after 8-9 h post infection. We observed a smooth
SIF tubule as shown in (Fig 3C see inset) which also appeared thinner. In the same
experiment, the fixative that is 3 % PFA was added to live cells on microscope stage and
images were taken after 10 min treatment. We observed that the Pseudo-SIF phenotype was
apparent in LAMP-1-GFP transfected cells after fixation. (Fig 3C. inset shows discreet
arrangement of LAMP-1 vesicles like Pseudo-SIF). We reasoned that SIF tubules are less
stable structures, and may also be disrupted by other drug treatments like nocodazole. To test
this, HeLa cells transfected and infected with sseF mutant was allowed to from SIF and 8 h
post infection, cells were taken for live imaging. Cells infected with mutant strains developed
SIF that were dynamic and showed extension and retraction of the tubule. (Fig 3D and Suppl.
movie 6). Later the cells were treated with 5g/ml nocodazole for 10 min, washed and again
imaged to see if Pseudo-SIF appear. However, the SIF seem to have the normal structures as
in non-treated cells expect that the dynamics was halted (Fig 3D). Therefore, from the above
experiments it could be concluded that SIF induced in sseF mutant infected cells have similar
properties as that of WT SIF, expect that they appear thinner than normal SIF and also depend
81
on microtubules for its dynamics. However the appearance of Pseudo-SIF can now be
attributed to the fixation artifact.
Fig. 3. Effect of fixation on the appearance of endosomal aggregates. A) And B) HeLa cells infected with WT
and sseF GFP and 12 h post infection, cells were fixed with 3 % PFA and processed for immunostaining. C)
Live cell setup was used as described for and LAMP-1-GFP-tranfected HeLa cells were infected with
Salmonella sseF mutant and post 8 h SIF were imaged (as shown in inset), later in the same experiment, 3 %
PFA was added to cells on microscope stage for 10 min and washed and imaged. D) Infected cells were imaged
at 8 h post infection prior and after addition of 5 g/ml nocodazole for 10 min and later washed and imaged.
Scale bar, 5m
These tubules induced by the sseF strain are somehow structurally unstable against fixation
and upon treatment with 3 % PFA lose their stability and appear like a punctuated
arrangement of LAMP-1 vesicles. Therefore from these studies we can say that fixation
causes artifacts on the organization of the endosomal system and care should be take for
interpretation.
82
The sseG strain
Intracellular phenotypes of the sseG strain were mostly identical to that of the sseF
strain. The formation of SIF with smaller diameter was observed, similar to the SIF induced
by the sseF strain. The dynamic properties of the SIF in cells infected with sseF or sseG
strains were indistinguishable (data not shown).
The sifA strain
This is one of the most studied mutant strains is the sifA strain as an important effector
of the SPI2 secretion system. Absence of SifA causes the loss of virulence in mice (Beuzon et
al., 2000) and many intracellular phenotypes have been reported that is caused due to lack of
SifA. One such notable phenotype is the absence of SIF formation in sifA mutant and loss of
SCV integrity during the course of infection. Mutants that lack SifA, is unable to maintain the
membranes around the SCV compartment and as a result SCV losses these membranes and
escape into the cytoplasm (Beuzon et al., 2000). When we were screening the mutants, we
reasoned that more detailed analysis is required in this aspect. We hypothesized that the SCV
compartment may still be intact in mutants lacking SifA and due to the fact that light
microscopes have limited resolution, we are not really able to see this during the normal
immunostaining procedure. Another reason was that fixation of samples might be subjected to
aberrant outcome as shown in case of the sseF strain. In order to study this, we set up live
imaging assays and also studied the phenotype by electron microscopy to get an in-depth view
of what is already reported.
A long time lapse movie was performed using the Spinning disc LSM microscope,
where images were taken every 5 min for a period of 1 to10 h post infection with the sifA
mutant strain in HeLa cells. As shown in (Fig 4A), there is lack of SIF in these cells but more
importantly (Fig 4B and Suppl. movie 7) highlights 2 SCVs which rapidly lose the LAMP-1
membranes around it towards 8 to 9 h post infection.
Detailed kinetics was also performed in live transfected infected cells to get an insight
into the kinetic of loss of SCV membrane in majority of infected cells (data not shown).
According to this, most of the SCV lose the LAMP-1 positive membrane around 7 to 9 h post
infection in contrast to previous reports which shows loss of SCV compartment as early as 4 h
post infection (Brumell et al., 2002). Therefore we can conclude that there is loss of SCV
membrane as reported previously but at late time point post infection. This point is further
supported by EM data which will be discussed later.
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SIF formation in sseF and pipB2 mutants infected RAW macrophages
It was previously shown that SIF formation is not only restricted to HeLa cells but are
also formed in macrophages (Both in RAW macrophages and primary peritoneal
macrophages) and dendritic cells (Rajashekar et al., 2008). To test the effect of SIF formation
in RAW macrophages by sseF and pipB2 mutants, RAW macrophages was infected with
respective mutants and treated with Alexa568-dextran to label the endosomes.
Fig. 4. Intracellular fate of a sifA strain in living host cells. A HeLa cells were transfected with LAMP-1-GFP and
infected with an S. typhimurium sifA strain constitutively expressing mCherry. Live cell imaging of infected cells
was started at 1 h post infection and time lapse series with intervals of 5 min were recorded over a period of 10 h
infection. B) And C) Note the disappearance of the LAMP-1-GFP positive signals around SCV 1 and SCV 2
respectively at 7 to 9 h post infection. Scale bar, 5m
Pulse chase experiments were performed as described in (Fig 5), after 8 to 9 h post
infection time lapse images were taken. As shown in (Fig 5 and Suppl. movie 8), SIF
formation was seen in all the strains without any difference in morphology as seen in HeLa
cells. Neither the bulky SIF of pipB2-infected cells nor the relatively thinner SIF of sseF-
infected cells were observed. Therefore mutants lacking the SseF or PipB2 proteins have no
effect on SIF formation in RAW macrophages as compared to that of HeLa cells.
84
Kinetics of formation of endosomal aggregations
Our previous analyses of the dynamics of SIF in HeLa cells infected with Salmonella
WT indicated that SIF extended in the early phase of intracellular life with a speed of
0.4m/sec, while the contraction was faster with 0.02m/sec (Rajashekar et al., 2008) as
analyzed by drawing kymographs. Here we investigated if the velocity of SIF extension and
collapse was affected by the function of effector proteins by again drawing kymographs as
shown in (Fig. 6).
Kymographs are time space plots and have been described in detail in our previous
work (Rajashekar et al., 2008). The sifA mutant strains were not further analyzed since tubular
endosomal aggregations were absent.
Fig. 5. Contribution of SPI2 effector proteins to SIF formation in macrophages. A) RAW macrophages were
infected with Salmonella WT or mutant strains defective in sseF or pipB2, each expressing GFP. Macrophages
were pulsed chased with Alexa568-dextran to label endocytic compartments. Live cell imaging was performed at
9 h post infection. Scale bar, 5m
The thin tubular aggregates induced by the sseF- or sseG-deficient strains also
exhibited highly dynamic characteristics similar to WT as shown in (Fig 6A & B). The
difference in peaks of the graphs represents the SIF extension and collapse and in turn shows
that there is constant growth and retraction. Thus SIF induced by sseF strain are highly
dynamic. In contrast, the bulky structures induced by the pipB2 strain (Fig 6C) have hardly
any dynamics as the graph is nearly liner, implying that SIF induced by the pipB2 strain have
nearly no growth or retraction compared to that of WT- or sseF-infected cells.
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Ultrastructural characterization of endosomal aggregations
The phenotypic differences in live cell studies of SIF induced by wild-type Salmonella
and mutants strains deficient in sifA, sseF, sseG or pipB2 also prompted us to investigate the
ultrastructure of SIF in infected HeLa cells. Analyses of a larger number of infected cells for
each mutant strain allowed us to identify SIF that were similar to what was seen in the light
microscope. The SIF of sseF mutant infected cells indeed had a very thin tubular structure as
seen on (Fig 7B) compared to WT infected cells which had normal SIF morphology (Fig 7A).
These EPON embedded plastic thin sections reconfirms our previous observation of SIF with
a thinner morphology. With regard to pipB2, as seen in (Fig 7C), thick bubble-like structure is
seen on EM sections depicted which could be attributed to bulky aggregates of endosomes as
seen in live cell assays.
Fig. 6. Kinetics of SIF extension and collapse in HeLa cells infected with Salmonella WT and various mutant
strains: HeLa cells were transfected with the LAMP-1-GFP construct and infected with Salmonella WT or the
sseF or pipB2 strains as indicated. At 6 h post infection, time lapse series of infected cells were recorded. SIF
86
were identified and the length of individual SIF was determined in 100 images of the time lapse series,
representing 500 msec time delay between each frame for a total time of 1 min. Kymographs was later drawn for
selected SIF using the EMBL Image J software. A) Kymographs drawn for WT induced SIF, which shows rapid
dynamics. B) Kymographs drawn for sseF-mutant induced SIF, where it also shows rapid dynamics. C)
Kymographs drawn for pipB2 induced bulky SIF, which is static without any peaks. Scale bar, 5m
In Fig 7D, the sections of next mutant under investigation can be seen, that is sifA. Though it
is clear that SIF are not induced by this mutant, it was mainly screened to see the loss of
endosomal membrane around the SCV at high resolution and which is also one the major
phenotype reported in sifA mutants (Beuzon et al., 2000). As shown in Fig7D, there is clear
loss of the membrane around the SCV. A couple of replicating bacteria is also seen to have
lost the SCV and is in the cytoplasm. It is however difficult to show statistically significant
data on the above phenotypes by this method, as this requires sampling of large number of
sections. Since sseF and pipB2 phenotype is found in merely 20 to 30 % of infected cells, this
is technically a challenging task. Thus to conclude this part, electron micrographs of WT, sseF,
pipB2 and sifA mutants respectively at high resolution reconfirmed the observations made by
the light microscopy assays.
Discussion
Live cell imaging allows tracing the fate of individual bacteria in infected host cells.
Live cell assays provide more reliable information of events taking place in cell compared
with previous studies in fixed cells. In this study we performed a detailed analysis of
contribution of SPI2 effectors to intracellular life of Salmonella in terms of SIF formation,
replication and SCV maintenance. Previously, several reports were published with respect to
phenotypes that arise from absence of SPI2 effectors. For example various phenotypes have
been shown for SPI2 effectors SifA, SseF, SseG, PipB2, SopD2, SpiC, etc (Guy et al., 2000;
Kuhle & Hensel, 2002; Jiang et al., 2004; Knodler & Steele-Mortimer, 2005; Boucrot et al.,
2005; Beuzon et al., 2000; Brumell et al., 2001; Stein et al., 1996). Formation of SIF is a
major hallmark of Salmonella infection and has been studied in detail by live microscopy in
previous studies (Rajashekar et al., 2008; Drecktrah et al., 2008). We extended this technique
to dissect the effect of various SPI2 effectors on SIF formation and dynamics.
87
Fig. 7. Ultrastuctural features of SIF induced by Salmonella WT and various mutant strains. HeLa cells were
infected with WT Salmonella or mutant strains lacking specific SPI2 effectors as indicated. The cells were
processed for EM analyses 10 h post infection. Representative cells for each infecting strain are shown. Scale
bar, 500nm
In this study we also investigated the effect of fixation as the major technique to study
intracellular phenotypes by immunostaining. As shown here, fixation causes the destruction of
fragile endosomal structures such as the SIF induced by sseF- or sseG-deficient strains. The
previously described Pseudo-SIF (Kuhle et al., 2002) thus may be the result of disruption of
thin tubular aggregates by fixation, resulting in the appearance of spherical membranes that
remain in the position of the previous SIF. Onset of SIF in cell infected with the sseF mutant
strain starts at a late time point post infection, and also there is moderate reduction of
intracellular replication of these mutant strains. Therefore, SseF secreted would be of
importance in early SIF formation and replication. Though there is certain amount of SIF
88
formation due to presence of SifA in mutants lacking of sseF or sseG, these effectors certainly
contributes to the complete formation and establishment for SIF as seen in WT-infected cells.
Other than the difference in appearance of SseF induced SIF, it could be noted that it hardly
had any effect on SIF dynamics. Also nocodazole treatment on cell infected with sseF mutant
had similar effect on SIF as compared to WT, which means that SIF require host microtubule
as scaffold for formation as shown previously (Rajashekar et al., 2008).
A further novel observation revealed by the live cell analyses was the unique structure
of endosomal aggregates observed in a fraction of the cells infected with the pipB2 strain.
PipB2 is previously shown required for the reorganization of LE/Lys during Salmonella
infection (Knodler et al., 2005). A simple over-expression of PipB2 changes the distribution
of vesicles and leads to redistribution of LE/Lys to the periphery.
However the mechanism responsible for this is not fully understood. It has been
reported by (Knodler et al., 2005) that PipB2 is required for the centrifugal extension of SIF
from the cell centre to the periphery. It has been proposed that the extension of SIF by PipB2
requires the microtubule plus-end motors and probably PipB2 modulates the net anterograde
movement of LE/Lys. Our study shows that a subset of pipB2-deficient strains shows the
aggregation of LAMP-1-GFP positive vesicles which we term as “Bulky SIF”. It could thus
be proposed that the occurrence of these structures may be due to lack of PipB2 to engage MT
plus end kinesin motors for extension of LAMP-1-positive tubules from cell centre to cell
periphery. However we also observed normal SIF morphology in another subset of cells in the
same experiment. The proportions of cells with bulky SIF and with normal SIF were almost
50% each (Data not shown). It is thus surprising how previous reports mainly have not
commented on this aspect (Knodler et al., 2005). As previously discussed, fixation and
immuno-staining method used by the authors might have disrupted these fragile structures
which in-turn might have caused shorter SIF. Revisiting the pipB2 phenotype in our study
only led to the finding of Bulky SIF, however the molecular basis of action of PipB2 in
LE/Lys positioning is still an open question.
Mutant strains defective on SifA were most severely attenuated in intracellular
replication as well as in systemic virulence, while mutant strains lacking either sseF or sseG
showed a moderate reduction of intracellular replication and systemic virulence in the mouse
model (Beuzon et al., 2000). Since SifA is central of SPI2-mediated virulence, our further
analysis of SifA reveled that mutants lacking sifA lose the membrane of the SCV and is
largely found in the cytoplasm as seen in live time lapse movie and in electron micrographs.
89
This is in accordance with the previous reports (Brumell et al., 2002), our EM studies shows a
clear loss of SCV membrane and number of replicating bacteria in the cytosol, indicating that
Salmonella has an edge over epithelial cells in establishing intracellular life. On similar
grounds SseF and PipB2 phenotypes were also reconfirmed by EM. Thus ultra structural
studies on mutants (sifA, sseF and pipB2) has given a strong ground for validating the
microscopic data which otherwise has a limited resolution. Another aspect is the analysis of
the above phenotypes in RAW macrophages. Many studies utilize epithelial cell lines to
examine intracellular phenotypes of S. enterica serovar Typhimurium. There could be genetic,
physiological or molecular difference in studying various cell types as model, like HeLa,
RAW macrophages, fibroblasts, CaCo-2 or MDCK cells.
Similarly, in our study we found that sseF-induced thin SIF and pipB2-induced bulky
SIF were absent when these strains were tested in RAW macrophages. Thus these phenotypes
are confined to HeLa cell culture models; however the significance of thin or bulky SIF (as
seen in sseF or pipB2) or SIF (as seen in WT) on the whole is not yet known. One of the
functions speculated for formation of SIF is to provide membrane source for the replicating
Salmonella in an expanding SCV or to provide other unknown host factors for establishment
of infection. This is not experimentally addressed till this date. It will thus be the focus of
future studies to assign functionality to SIF which would then be of interest to probe
contribution of effectors to SIF function in context to outcome of Salmonella infections.
Materials and Methods
Bacterial strains and growth condition
Salmonella enterica serovar Typhimurium NCTC12023 was used as the wild-type
strain and all mutant strains were isogenic to this strain. For certain experiments, mutant
alleles were transduced into strain background LT2A. We have previously compared
intracellular phenotypes of wild-type strains NCTC12023 and LT2A and did not observe
differences in the induction of SIF (Rajashekar et al., 2008). The construction of mutant
strains defective in single effectors has been reported before and the characteristics of the
strains are listed in the table 1 (Suppl. section – Table 1). Further strains defective in defined
SPI2 effector genes were generated by the ‘one step inactivation’ approach basically as
described before. For complementation of mutations in genes encoding effectors, low copy
90
number vectors are constructed for the expression of the effector proteins under control of
their own promoter.
Cell Culture
Human epithelial cell line HeLa cells (ATCC No CCL-2) were cultured in DMEM
with 10 % FCS, penicillin and streptomycin, and grown in 37°C in a humidified atmosphere
containing 5 % CO2. Other cell line used in this study was murine macrophage like cell line
RAW 264.7(ATCC No. TIB-71), which was also grown in the above specified manner. For
infection of epithelial cells, all the strains were grown in LB broth with carbenicillin
100g/ml for overnight and sub-cultured for 31/2 h (till it reaches late log phase) and then
used for infection at MOI of 50. For infection of Raw, firstly cells were stimulated with 5ng
IFN /ml for 16 to 18h, which resulted in about 90 %, activated macrophages as judged from
the cell morphology. Later the strains were grown overnight (till it has reached stationary
phase), and then directly used for infection at an MOI of 50.
Transfection and infection of HeLa cells
HeLa cells (about 2 x 104 cells) were seeded in wells of an 8 chamber glass slide
(Nunc-Labtek) and allowed to adhere overnight. About 500g of plasmid DNA (LAMP-1-
GFP kindly provided by Patrice Bouquet) were mixed with the transfection reagent and added
to cells in 8 chamber slide with DMEM with 10 % FCS. Cells were incubated for 4 to 5 h,
then medium was changed and fresh DMEM with 10 % FCS was added. Cells were used for
infection studies 16 to 18 h after transfection. For infection, S. typhimurium was sub-cultured
for 3.5 h and the culture was diluted to an OD600 of 0.2. Cells were infected at a multiplicity
of infection of 50 and infection was allowed for a period of 30 min. Subsequently, non-
internalized bacteria were removed by washing thrice with PBS and then DMEM containing
10 % FCS and gentamicin at 100g x ml-1 was added to kill remaining extracellular bacteria.
After incubation for 1 h, the cell were washed again and imaging medium (Imaging medium
is Eagles Minimum Essential Medium (MEM) without L-glutamine, phenol red and sodium
bicarbonate, containing 30 mM HEPES, pH 7.4) containing 10g x ml-1 gentamicin was used
for the rest of the infection. The chamber slide was then taken for imaging at required time
points after infection. The chamber slide was mounted on the microscope stage equipped with
an incubation chamber maintaining 37°C and 5 % CO2.
91
Fluid phase marker and pulse chase
For tracing the endocytic pathway, fluid phase markers were used, HeLa cells were
transfected with LAMP-1-GFP, infected as described and 4 h post infection, cells were
incubated with 100μg x ml-1 Alexa568-dextran, (molecular weight 10,000, Molecular Probes)
for 3 h, washed, and incubated for the rest of the experiment with dextran-free media. Later
cells were processed for imaging. For raw macrophages, cells were infected and after 6 to 7 h
post infection, cells were incubated with 50μg x ml-1 Alexa568-dextran (molecular weight
10,000, Molecular Probes) for 30 min, cells were washed thrice with PBS and then incubated
with DMEM with 10 % FCS and chased for 1 h (until the endocytosed dextran accumulated
in late endosomes and lysosomes) and then processed for imaging.
Immuno-staining and Nocodazole treatment
HeLa cells (non-transfected) were infected with various Salmonella strains expressing
GFP as described above and after 12 to 16 h post infection, cells were fixed with 3% PFA for
10 min. and washed and stained with primary anti-mouse LAMP-1(1:250) for 1 h, washed
with PBS 3 times and secondary staining was performed using goat anti mouse Cy3 for 45
min.
For nocodazole treatment, HeLa cells transfected with LAMP-1-GFP were infected with
various Salmonella strains and 8 h post infection, cells were treated with 5µg x ml-1 of
Nocodazole for 30 min., washed three times with PBS and imaged using a Zeiss Axiovert
200M wide field microscope.
Microscopy and imaging
Live cell imaging was performed basically as described before (Rajashekar et al.,
2008). Imaging studies were done using the Perkin Elmer spinning disc confocal microscope
(UltraVIEWRS-ERS) mounted on a Zeiss Axiovert 200 microscope with an acoustic optical
tunable filter for wavelength selection (ATOF) for 4D image acquisitions. The microscope is
equipped with a highly sensitive CCD camera, and 5 laser lines along with Nipkow spinning
disc for high temporal acquisition with stacks or 4D imaging. Image acquisitions were
performed at different time points using a x100 Plan Neofluar objective at various time points
post infection and images stored in a given data format. Live imaging at lower temporal
resolution was performed using the Zeiss Axiovert 200M confocal microscope equipped with
wide field illumination and an AxioCam MR camera.
92
The resulting movie series were corrected for background fluorescence and bleaching using
bleach correction and background subtraction macros available at EMBL Image J (available at
http://www.embl.de/almf/html/downloads.html). Majority of the image analysis (to calculate
the velocity of SIF growing and shrinking was done using the macro Kymograph written by
Arne Seitz at EMBL Image J (Seitz & Surrey et al., 2006 also the detailed generation of
kymograph is explained in our previous study Rajashekar et al., 2008).
For long time lapse series, HeLa cells were transfected and infected with sifA mCherry
strains as described above and after 2 h post infection, the cells were taken for imaging with
the Perkin Elmer RS. The time lapse parameters include taking every image between 5mins
interval for total time duration of 10 h.
Electron microscopy
For plastic (EPON) sections: HeLa cells were grown on glass cover slips in plastic
culture dishes in complete DMEM medium. Cells were infected with S. typhimurium at MOI
= 50 and incubated for 10 h prior fixation. All samples were fixed 10 h post infection in 2 %
glutaraldehyde in 0.1 M cacodylate buffer (CB), post-fixed in 2 % osmium teroxide,
dehydrated in ethanol series and propylenoxide, flat-embedded and polymerized in EPON812
(Serva). Glass coverslips were removed in liquid nitrogen and blocks with cell monolayer
were embedded and polymerized again to enable cutting in parallel or perpendicular to Z axis.
Sections (40 nm) were cut with diamond knife on Reichert Ultracut S and contrasted in uranyl
acetate and lead citrate. Samples of plastic sections were observed with a Phillips Morgagni
EM 268D operating at 100 kV. Pictures were taken using CCD camera and AnalySIS 3.2
software. Final Figs were edited with Adobe Photoshop 8.0.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft as project C1 in SFB 796.
We like to thank Arne Seitz for support of this work at the ALMF of the EMBL, Heidelberg.
Abbreviation list
MT, microtubule; SIF, Salmonella-induced filaments; SPI1, Salmonella Pathogenicity Island
1; SPI2, Salmonella Pathogenicity Island 2; SCV, Salmonella-containing vacuole, T3SS, type
III secretion system WT, wild type.
93
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Supplementary Material
Novel functions of SPI2 effector proteins during intracellular pathogenesis of Salmonella
enterica revealed by live cell and ultrastructural analyses
Roopa Rajashekar1, David Liebl2, Deepak Chikkaballi1 and Michael Hensel1
Mikrobiologisches Institut, Universitätsklinikum Erlangen1, Cell Biology and
Biophysics Unit, EMBL Heidelberg2
Movie 1 Corresponding to Fig 1: Time lapse movies of all the SPI2 effectors which is shown
in gallery. This set of movies shows an overview of SIF morphology and dynamics of
respective effectors. Scale bar, 5 m.
Movie 2 Corresponding to Fig 2A: This movie shows the SIF induced by WT Salmonella
post 8h infection. Scale bar, 5 m.
Movie 3 Corresponding to Fig 2B: This movie shows the SIF induced by sseF mutant post 8h
infection. Note: the SIF that appear thin compared to WT. Scale bar, 5 m.
Movie 4 Corresponding to Fig 2C: This movie shows the SIF induced by pipB2 mutant post
8h infection. Note the SIF that appear “Bulky” termed “Bulky SIF” compared to WT. Scale
bar, 5 m.
96
Movie 5 Corresponding to Fig 2E: This movie shows the SIF induced by sseF and pipB2
mutant post 8h infection, and complimented by appropriate plasmid and the phenotypes are
restored. Scale bar, 5 m.
Movie 6 Corresponding to Fig 3D: This movie shows the dynamic SIF induced by sseF
mutant and non dynamic SIF after nocodazole treatment. Note: the SIF structure is not
disturbed in nocodazole treated cells. Scale bar, 5 m.
Movie 7 Corresponding to Fig 4: This is the 1 to 10h long time lapse movie of HeLa cells
infected with sifA mutant. Note: at the bottom of the movie appear the highlighted SCVs that
lose the LAMP1-GFP marker. Scale bar, 5 m.
Movie 8 Corresponding to Fig 5: Interferon -stimulated RAW264.7 macrophages were
infected with Salmonella WT, sseF or pipB2 mutant strains expressing GFP. The cells were
pulsed with Alexa568-dextran 5 h post infection and imaged 2 h later. Note: there is no
difference in the appearance of SIF in mutants infected cells compared to WT. Scale bar, 5
m.
Tables
Table 1. Strains and plasmids used in this study
Strains Genotype, relevant characteristics Reference
NCTC 12023 Wild type lab collection
MvP915 ∆sseC::aph this study
MvP912 ∆sifA::aph this study
MvP916 ∆sifB::aph this study
MvP910 ∆sseF::aph this study
MvP911 ∆sseG::aph this study
MvP917 ∆sseJ this study
MvP914 ∆pipB2 this study
97
MvP913 ∆sopD2 this study
MvP379 ∆slrP (Chakravortty et al., 2002)
MvP375 ∆sseI (Chakravortty et al., 2002)
MvP376 ∆sspH1 (Chakravortty et al., 2002)
MvP378 ∆sspH2 (Chakravortty et al., 2002)
MvP741 ∆steC this study
MvP1033 ∆sseL this study
MvP570 ∆sseK1 (Chakravortty et al., 2002)
MvP571 ∆sseK2 (Chakravortty et al., 2002)
MvP874 ∆pipB (Chakravortty et al., 2002)
EG10128 ∆spiC this study
MvP873 ∆gogB Halici
MvP1208 ∆sopB::aph this study
MvP450 ∆sseJ sifA::mTn5 this study
98
Chapter-3
3.3 Ultrastructural analysis of SCV and Biogenesis of SIF by Electron
tomography
Introduction
Electron microscopy is a technique that is used to visualize objects that are as small as
1 nm or below. The basic principle of EM involves scattering of electrons which is the source
use to illuminate the specimen. Electron microscopy technique can be broadly classified to
transmission electron microscopy (TEM) and scanning electron microscopy (SEM). TEM is
used to study the inner structure of objects (tissues, cells, viruses) etc and SEM is used to
visualize surface of tissues and macromolecular complexes. Recent developments in
microelectronics and data processing have made transmission electron microscopy (TEM) a
powerful tool for the study of whole organelles and cells. Thus EM is a technique that
combines sensitive protein-detection methods and structure information to better understand
the cellular processes. (Reviewed in Vladan Luˇci´c, et al., 2005)
Transmission Electron Microscope (TEM)
During Transmission electron microscopy (TEM) a high voltage electron beam
emitted by a cathode is condensed and focused by electro-magnetic lenses. The electron beam
that passes through the specimen produces the image which is magnified by a series of
magnetic lenses which is then recorded by a CCD camera and displayed on monitor. There are
various steps to TEM from sample preparation, to image acquisition and interpretation
(Reviewed in Vladan Luˇci´c, et al., 2005). Since discussion of all these aspects are beyond
the scope of this section, more emphasis will be on the application of this method in cell
biology as a whole and in particular to address our scientific problem.
Applications of Electron Tomography in SCV and SIF Biogenesis
Electron tomography has a wide range of applications in cell biology from studying
the cells, organelles to different membrane organizations to viewing whole cells like viruses
and bacteria (Reviewed in Vladan Luˇci´c, et al., 2005). The following section throws light on
the application of EM/ET to this work. It also addresses why we chose this method or
technique to describe the SIF and SCV.
In previous studies, various approaches have been taken to describe SIF and SCV mainly by
immuno-staining, live cell imaging and also electron microscopy.
99
Though these techniques were of enormous help in providing insight into various
phenotypes of intracellular Salmonella, the spatiotemporal arrangement of the SCV and SIF
within host cellular space and environment was not clear. There were several studies where,
ultrastructure on Salmonella infected cell were performed to show that for example SCV were
disrupted due to lack of SifA. Here by using EM the authors have shown the presence of SCV
in cytoplasm of cell, thus claiming that SifA is responsible for maintaining SCV integration
(integrity) (Beuzón et al., 2000). Another example employed scanning electron microscopy to
reveal novel surface structures of SPI2 translocon of Salmonella (Chakravortty et al., 2005).
The event of invasion with ruffle formation in epithelial cells or intracellular membrane
bound SCV was shown by EM (reviewed in Brumell et al., 2000). However, only few studies
described in detail the membrane organization of SCV and SIF during the course of infection.
Previous work and our observations of SCV interaction with the endosomal pathway and its
remodeling (Drecktrah et al., 2007, Rajashekar et al., 2008) was very intriguing which lead us
to ask many questions such as:
a) How is the membrane organization process taking place and what are its effects on
biogenesis of SIF and other host membrane compartments?
b) What is the nature of SIF structure at ultrastructure level?
ET/EM has been used by many studies to elaborate on the spatial arrangement of the
pathogen within the host, or to study fine structures of the pathogens itself. For example, the
study of Tomoval and group (Tomoval et al., 2009) has described using EM/ET and high
pressure freezing (HPF) preservation techniques to elaborate on Toxoplasma gondii apicoplast
membrane organization, its association with Endoplasmic Reticulum (ER). The finding that
there is no fusion or continuity between the ER and the apicoplast membranes has a major
input in understanding the protein and lipid trafficking through the endosomal membrane
system to the apicoplast. ET and HPF/FS (freeze substitution) was the choice here due to the
fact that membrane structure was always destroyed by other conventional methods according
to the authors. Another example where ET has been very useful is in studying the structure of
mycobacterial cell wall (Hoffmann et al., 2008).
Though there were reports that mycobacterial cell wall is a complex structure of lipids,
it was not proven experimentally with clear structure of cell wall and its membranes. In this
study it was for the first time shown that mycobacterial outer membrane is a lipid bilayer and
is symmetrical. This was not possible with previous studies as the structure was distorted by
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fixation. The authors say that the near to life state of preservation (using cyropreservation
technique) allowed the understanding of this complex structure. All these studies have thrown
light on the importance of EM/ET in basic understanding of membrane structures due to
advancement in preservation techniques and provision of generating 3 dimensional models.
Purpose of choice of EM and ET for our current work
As discussed above, although some groups have used EM for describing Salmonella
phenotypes, none have addressed in detail the membrane organization of SCV and SIF with
respect to its host. Here for the first time we have reported the ultrastructure of SIF and SCV
on ultra-thin structures which was published (Rajashekar et al., 2008). During the course of
this work we also came across puzzling findings which are discussed later, that prompted us
to continue with this work. We then also sought to understand the biogenesis of SIF through
the course of infection using EM/ET technique.
The choice of EM/ET for this work was to get a clear understanding of the membrane
organization of the SIF and SCV with respect to host in the given microenvironment. As
discussed above, though normal convectional method of chemical fixation and ultra-thin
structures were initially generated to study the gross morphology of SIF, these sections still
did not give a clear idea of spatial arrangement of complex membrane structures that we often
saw. This feature was attributed to inefficient preservation of the structures in the native state.
We needed samples with a reliable preservation of the membrane compounds in their
three dimensions. A preservation that will guarantee that all constituents of the cell are
immobilized before significant rearrangement can occur. Appropriate fixation is of crucial
importance especially for an organelle such as the SCV and SIF. Therefore we employed High
pressure freezing and freeze substitution (HPF/FS) where Salmonella infected samples are
subjected to freezing at low temperatures and then further the samples are substituted with
organic solvents at low temperatures by a slow process by eliminating water crystals which
could otherwise cause damage to the biological structures. This method is artifact-free, where
high-resolution images can be taken for obtaining three dimensional information of a
biological structure.
Therefore, specimen preparation by HPF, followed by FS (Marsh et al., 2001) and resin
embedment ensures structural preservation as close as possible to the living state. It has been
proven that the three-dimensional arrangement of membranes is retained and only few lipids
are lost (Kremer et al., 1996, Smith et al., 1999), in addition, this approach gives the
possibility to examine large sample areas. The combination of HPF/FS sample preparation
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and ET proved to reveal not only the morphology of cellular structures in its native context
but also the relationships they have at high resolution (Szczesny et al., 1996). This changed
our understanding of complex membranous structures and organelles in the cell, their spatial
connections and functionality.
Results
Ultrastructure of SIF reveals SIF with double delimiting and single delimiting
membranes
In order to follow the ultrastructure of SIF/SCV by TEM, we first used standard
sample processing for plastic (EPON) embedding. Initially we detected two types of SIF-like
structures in number of thin sections analyzed. We therefore thought that two subpopulations
of SIF coexist. This is shown in Fig 1A; where one subpopulation of SIF is described as long
tubule with a double delimiting membrane with the cytoplasmic content. This bilayer
structure was a surprising finding as very few organelles in the cell (expect plasma membrane
and mitochondria) have double membrane structures. In order to explain the double
membrane of SIF, We performed control experiments with mitotracker to see if there is any
colocalization of mitochondria with SIF (Data not shown). However we did not find any
colocalization of mitochondria with the SIF. Neither ER nor mitochondria was found in close
proximity with the double membrane SIF on ultrathin sections. The second subpopulation was
seen more often and we detected SIF densely packed with lysosomal content and delimited by
a single membrane (Fig 1B). The co-existence of these two morphologically distinct types of
SIF is explained in the model we have proposed (will be discussed later in this section) based
on our observations from electron microscopy analysis and live cell imaging.
Branching of SIF
Branching and network of SIF as seen in the live cell assays (Fig 2B) was one of the
observations where single dynamic SIF during the course of infection undergo remodeling
due to constant fusion of endosomal vesicles which later leads to branching and several SIF
thus connect to become one large SIF network which are usually static (Rajashekar et al.,
2008).
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Fig 1: Double membrane SIF and single membrane SIF: A) HeLa cells were transfected with LAMP-1 and
infected with WT Salmonella and after 10 h post infection, cells were fixed and processed for EM (as given in
material and methods). A) Tomogram tilt series with showing double delimiting SIF with cytoplasmic contents.
B) Tomogram showing SIF with a single delimiting membrane with lysosomal contents. Scale bar, 500nm
At the ultrastructural level the acquisition frequency of longitudinal cross-sections through the
SIF tubule along its whole length is relatively rare. This is given by the fact that single ultra-
thin section represents only about 1% of total volume of the cell and is rarely in complete
parallel to the SIF tubules. Moreover, SIF tubules are often curved in space and thus often go
Double delimiting membrane
SCV
A B
Single delimiting
membrane
Double delimiting
membrane
Double delimiting membrane
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out of the plane of the thin section. But though the SIF branching was a rare event seen in
EM, it is however shown also here and this reconfirms our live cell observation that SIF are
complex network of membranes. As seen in Fig 2A, SIF are branching with both
subpopulations of SIF double and single delimiting membranes intertwining in the network.
Fig 2: Branching of SIF: HeLa cells were transfected with LAMP-1 and infected with WT Salmonella and after
10 h post infection, cells were fixed and processed for EM (as given in material and methods). In A) both SIF
subpopulations are seen branching. Scare bar 500nm. B) Comparison of SIF network seen in Salmonella infected
cell 8 h post infection as seen in live cells (also refer to Chap 1 Fig 7). Scale bar, 5m
Cytoskeletal Structures inside SIF Lumen
In yet another surprising finding, we saw that cytoskeletal elements like microtubule and actin
filaments were found wrapped inside the SIF lumen. This is shown in (Fig 3) where SIF
lumen contains cytoplasmic contents along with microtubule (MT) (Fig 3A) and actin
filaments (Fig 3B). Therefore it is clear that SIF enwrap the cytoplasmic contents including
cytoskeletal structures. We could thus speculate that due to the presence of these cytoskeletal
structures inside SIF lumen, SIF gain stability at later time point post infection and thus
establish a network and cease its dynamic nature.
A B
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Fig 3: HeLa cells were transfected and infected with Salmonella WT, and post 10 h post infection, cells were
processed for EM and thin EPON sections were cut and analyzed. The above sections show A) SIF with a double
delimiting membrane and microtubule (MT) inside the lumen of SIF. B) SIF with a double delimiting membrane
with actin filaments inside the lumen of SIF. Scale bar, 200 nm
HPF-FS and its impact on structural preservation of Salmonella, SCV and SIF
membranes
The classical chemical fixation protocol followed by dehydration and plastic flat-embedding
provided relatively good ultrastructural preservation of cell structures so as of Salmonella
within the SCV. In contrast to majority of membranous structures of the host cell the double
layer of the SCV membrane was not always intact. Dashed-line of high-contrast membrane
suggested possible perforations and that could be linked to membrane pore formation (data
not shown) induced by Salmonella. Interestingly, a high-resolution inspection of these
membrane in-continuities revealed that rather than membrane pores they represented patches
of membrane bilayer with extremely low contrast in comparison to surrounding membrane
domains.
To test whether the ultrastructural characteristic of SCV membrane was not an artifact
of sample processing, we set up HPF-FS as an alternative processing method that provides
superior structural preservation in comparison to the conventional processing. During HPF,
Actin Filaments Microtubules
A B
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the cell monolayer can be rapidly frozen (vitrified) within milliseconds by a jet of liquid
nitrogen at a high pressure that prevents ultrastructural damage induced by ice crystal
formation. The cellular water is then slowly exchanged for organic solvent (acetone)
containing fixative and/or contrasting agent (Osmium tetroxide or Uranyl Acetate). This
exchange is carried out at very low temperatures (-90 to -30°C) which minimizes the artifacts
caused by conventional fixation and dehydration at room temperature.
Comparison between these two methods of sample preparation is shown in the (Fig 4).
Here we focus on comparison of the Salmonella inner and outer membranes and the
membrane of the SCV. The results show a great advancement in ultrastructural preservation
of all cellular structures including Salmonella.
Fig 4: Comparison of structure preservation: HeLa cells were transfected with LAMP-1 and infected with WT
Salmonella and after 10 h post infection, cells were processed for EM by conventional chemical fixation,
dehydration and embedding at RT (upper panels) or by HPF-FS (lower panel). Note the discontinuities in SCV
membrane and shrinkage of Salmonella membranes (upper panels) in comparison to smooth, intact and continual
membranes with homogenous spacing of distinct leaflets of membrane bilayer (lower panels). Scale bar, 200nm,
inset: 20nm
This allowed us to distinguish individual leaflets of two Salmonella membrane
double-layers including periplasmic layer in between and adjacent double-layer of the SCV in
a high resolution. Therefore to conclude this part some of the previous observations from
conventional preparation where low contrasted membrane structures believed to be pores
were not found with HPF-FS method of sample preparation. Thus these structures may be
106
artifacts caused due to chemical fixation. This method was mainly used to prepare samples for
electron tomography studies.
Ultrastructure of SIF in RAW macrophages
In our previous observations, we have shown that SIF are not only limited to HeLa
cells but are also found in RAW macrophages (Rajashekar et al., 2008). We therefore wanted
to investigate if there is any structural difference between SIF in HeLa cells and SIF formed
in RAW macrophages. We were also interested whether the SIF and/or SCV are in continuous
contact with endosomal/lysosomal system.
To test this, we loaded the cells with BSA-gold as a fluid phase marker and analyzed
the thin sections for a presence of BSA-gold in the lumen of SIF/SCV (Fig 5). In RAW
macrophages, a very complex SCV with network of membranes was seen which appears
much more complicated than in HeLa cells. Here also double membrane SIF like structures
were seen with the surrounding cytoplasmic contents. Most importantly we could also
observe gold particles in the lumen of SIF (Fig 5 see right panel enlarged inset) which is in
agreement with our previous observation by live imaging that fluid markers get access to
these compartments. This assay also confirmed that the SCV is in a constant interaction with
endocytic pathway. From these experiments it can be concluded that SIF are present in both
HeLa and RAW macrophages though SIF are morphologically more complex in RAW
macrophages compared to HeLa cells. However Salmonella induces these structures in both
cell culture models under study.
3D rendering of SIF by EM tomography
One of major aim of this work was to generate 3 dimensional images of SIF in order
to get in-depth view on SIF structure, membrane organization, and its spatial arrangement
with respect to SCV as well as to learn more about biogenesis. For this electron tomography
was performed on thick section of HeLa cells infected with WT Salmonella (see materials and
methods for more details). Here samples generated by HPF/FS were subjected to tomography
where digital images (bin 2, 2048 x 2048) were taken every 1°, over a ± 60°- 65° range with a
pixel size ranging from 0.5-1 nm using Serial EM acquisition software. Fig 6 displays the 3D
rendered structure of SIF, which shows the appearance of SIF in volume. As shown in Fig 6,
it is now easy to understand SIF organization with respect to SCV. We can now imagine the
hollow tube of SIF with its external and internal lumen and also gold loaded fluid phase
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markers in the lumen of SIF which is accessible to SCV. This and other tomograms helped us
to propose model of SIF biogenesis which will be discussed in the following sections.
Linking the SIF/SCV ultrastructure with model of their biogenesis
Ultrastructural and electron tomography analysis of SIF and SCV lead us to propose a
model for SIF biogenesis. SIF formed over a time of infection undergo temporal and
morphological changes in its structure. Our initial analysis of SIF by EM gave us some
puzzling observations, where we found that SIF are double membrane structures. Therefore it
was very important to explain this phenomenon and thus we further generated tomograms and
analyzed to find that there are two types of SIF subpopulation, SIF with double membranes
and SIF with single membranes.
Fig 5: SIF in HeLa and RAW macrophages: HeLa cells and RAW macrophages were infected with WT
Salmonella and pulse-chased with 10nm BSA gold (as specified in materials and methods) and after 10 h post
infection, cells were processed for EM by HPF-FS .In the above picture, left panel shows SIF in HeLa cells
(Note both types of SIF subpopulations) and the right panel shows SIF in RAW macrophages (note both the
double membrane as well as gold-loaded lumen of SIF (boxed region -see inset with the arrow). Scale bar, 1m
We now propose that double membrane SIF originate from single membrane SIF by
“Spontaneous Membrane Reorganization”. This can be explained by the fact that there is
certain threshold of surface to volume ratio of SIF/SCV membranes and the continuous
process of endosomal fusion delivering its contents. We then ask the question as to what
happens when surface to volume ratio increases and/or membrane surface tension relaxes over
HeLa Cells RAW Macrophages
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a certain threshold? We propose that the excess of surface membrane in proportion to the
luminal volume of SCV/SIF can induce membrane folding on the SCV/SIF surface which
readily results in membrane invagination or exvagination within the SCV/SIF. This model of
membrane folding and organelle flattening is supported by our observations of cytosol-
containing “bubbles” within the SCV/SIF which we interpret as cross- or longitudinal section
through invaginations of SCV/SIF surface membrane to the SCV/SIF interior space (Fig. 7).
Fig 6: 3D rendered SIF: Tomograms were generated using thick sections of HeLa cells infected with WT
Salmonella. The above picture shows the SCV and SIF in 3D volume where the green contour is the SCV and
orange and yellow contour are the inner lumen and outer membrane respectively.
Nevertheless we show that without large 3D-volume reconstruction it is difficult to specify
whether the membrane delimited cytoplasm-containing “bubbles” or “pockets” represent only
deep membrane invaginations from the SCV surface or can be completely enwrapped within
the SCV being thus isolated.
The latter would require the fusion of membrane above the invagination – a process
topologically similar to formation of intraluminal vesicles in sorting endosome from SCV/SIF
compartment can develop into a complex 3D structure, where the surface membrane contains
Salmonella within a space that is clearly continual with the lumen of lysosomes (See electron-
dense lysosomal granules and endocytosed BSA-gold nanoparticles on Fig.8). Ultrathin EM
SIF inner membrane (Orange contour)
Outer membrane
(Yellow contour)
BSA Gold particles in the lumen of SIF (Black contour)
Salmonella (Green contour)
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sections through the SCV/SIF also show how invaginations of SCV/SIF surface membrane
and/or SCV/SIF flattening brings folded membranes in a close contact with each other what
substantially reduces the local luminal volume.
Fig 7: A) HeLa cells are infected with WT Salmonella and 10 h post infection samples are processed for EM.
Cross section of a tomogram showing SIF invaginations of SCV/SIF surface membrane to the SCV/SIF interior
space. This also shows the cytoplasmic bubble enwrapped in SCV/SIF membranes. Scale bar, 2m
As a result, the dense lysosomal content is locally squeezed aside within the SIF
lumen wherever the membrane folding occurs (Fig. 9A and model on Fig. 9B). 3D rendering
of tomogram also explains this model very clearly (Fig. 9C). The rendered tomogram makes it
clear to visualize the cytoplasmic bubbles trapped within the SCV invaginations. This model
explains why SIF that appear on ultrathin sections have sometimes features of tubular
lysosome or they are delimited by two membranes with rather empty lumen in between. We
show that these distinct characteristics do not reflect separate populations but dynamic
developmental stages of individual SIF.
Furthermore, this ultrastructural analysis explains our live imaging observations of
leading vs. trailing SIF (Rajashekar et al., 2008) where the thick (estimated by fluorescence
intensity) portion of SIF tubule seemed to move bi-directionally within the thin SIF with a
kinetics reminiscent of intestine peristaltic. Here we propose that the trailing SIF likely
represents the cytoplasm-containing “bubble” invagination being also physically thicker
(estimated from a diameter on EM images). Conversely, the leading SIF would then correlate
with the portion of SIF filled with a dense lysosomal content and delimited by a single
membrane. Thus Salmonella is initially in a direct contact with the lysosomal content within
Red contour shows the
SCV/SIF invaginations
Enwrapped cytoplasmic
bubbles in yellow contour
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the SCV. However, extensive membrane folding and surface membrane invaginations may in
turn result in partial/local isolation of the bacteria.
Fig 8: HeLa cells are infected with WT Salmonella and 10 h post infection samples are processed for EM. This
tomogram shows cytoplasm-containing “bubbles” or “pockets” which represent only deep membrane
invaginations from the SCV surface. Scale bar, 1m
Discussion
In this study, we set out to understand the basic ultrastructure of SIF and SCV in the
context of other membrane compartments of the host cell after infection with Salmonella. We
initially detected and described two types of tubular membranous structures that we correlated
to SIF. However, our further analysis revealed that both, the tubular lysosome-like SIF with a
lumen densely packed with lysosomal granules and the thicker SIF that reminds a flattened
membrane cistern wrapped into a hollow tube are derived from the same SCV. We show that
although these SIF seem to be morphologically distinct, they both represent longitudinal
cross-sections through membrane extension of the same SCV with a high 3D complexity. Our
previous analysis of SIF dynamic by live imaging have shown that SIF tubules undergo
extensive remodeling that is linked with their branching and kinetic properties (Rajashekar et
al., 2008).
Electron dense BSA
gold nanoparticles
Contour in red represents SCV
Contour in yellow represents
the cytoplasmic bubbles inside
the SCV lumen
Salmonella
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Fig 9: HeLa cells are infected with WT Salmonella and 10 h post infection samples are processed for EM. A)
Tomogram showing SCV and SIF membrane enwrapping the cytoplasmic bubble (Red counter). B) Cartoon
explains the model SIF have features of tubular lysosome or they are delimited by two membranes with rather
empty lumen due to addition of membranes and this results in membrane flattening and wrapping of cytoplasmic
contents. C) This is a series of double tilt rendered tomogram which shows SCV membrane invaginations which
create open and closed "bubbles" of cytoplasm. The lysosomal content is in brown (based on electron-dense
threshold filtering), SL in green, SCV membrane in yellow. Scale bar, 1m
Here we suggest that this inter-changeable ultrastructural characteristic of SIF is a
result of spatio-temporal remodeling of the excess membrane (folding, extension, wrapping
etc.) that cumulates on the SCV as a result of continuous fusion with the endocytic vesicles.
From the mechanistic view, all membrane fusion/fission processes within cells result in dis-
balance of surface to volume ratio and/or in changes of membrane surface tension on the
resulting organelle or membrane vesicle.
A
C
B
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Modulation of transient changes in membrane properties is tightly dependent on
protein and lipid composition and their spatio-temporal distribution, but how these processes
are regulated in cells is still poorly understood. Based on our ultrastructural analysis, electron
tomography and kinetic studies using live cell imaging we designed a model of SCV/SIF
biogenesis whereby the continuous and excessive fusion of endosomes with the SCV
eventually leads to substantial changes in its surface to volume ratio. We suggest that at early
stages of SCV/SIF biogenesis (6-9 h p.i) their surface to volume ratio and membrane tension
is initially balanced by dynamic SIF elongation into 3D tubular membrane network with
relatively large surface. However, at later stages (from 10 h p.i) the SIF loose their dynamic
properties and a stable network is established with minimal ongoing net growth. Here we
imply that further fusion of endosomes with the membrane of static SIF network will
inevitably increase the surface to volume ratio and relax the existing membrane tension.
Similar scenario can most likely occur in the early stages as well if the fusion of endosomes
with the SCV/SIF is locally and/or temporarily) faster than the rate of SIF extension.
The next aspect was that SIF exhibited a more complex structure in RAW
macrophages then in HeLa cells. This could be attributed due to different background of these
cell culture models. Also it should be noted that RAW macrophages are morphologically
small cells compared to HeLa cells and we also activate them using the IFN. Due to this fact
RAW macrophages undergo gross morphological changes with highly tubular endosomes
seen even before Salmonella infection (Rajashekar et al., 2008). This condition may add more
complexity to the SCV and membrane organization in these cells as a whole. We confirmed
that fluid phase markers could reach the luminal space of SIF/SCV and surround the
Salmonella together with the granular, electron dense material typical for content of
lysosomes and late endosomes. It has been originally suggested in literature (Garcia-del
Portillo et al., 1995; Rathman et al., 1995; Garvis et al., 2001) that Salmonella actively blocks
fusion of SCV with endo-lysosomal system; however this has been recently disproved by
several studies (Drecktrah et al., 2007, Rajashekar et al., 2008). Although our results bring
another evidence for continuous fusion of the SCV with LE/LY, we also show that the
membrane invaginations and their folding within the SCV can at least partially shed
Salmonella from physical contact with lysosomal content.
The function of SIF is unclear and has not been experimentally proven until now. It is
speculated to be important for replication (Garcia-del Portillo et al., 1993); however the
experimental evidence is missing. In our previous work on SIF and its dynamics we
speculated that the purpose of SIF formation might provide constant supply of nutrients to the
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rapidly replicating Salmonella within the SCV. However from the observations from this
study we can speculate that continual fusion of SCV with endosomes brings nutrients for
replicating Salmonella within SCV, but not the SIF. The BSA-gold experiment supports this
view because these nanoparticles end up in the lumen of the SIF and SCV. Then the question
would be is why the growing SCV does not stay like spherical vacuole but extends into
tubular SCV instead?
Finally the advanced electron microscopy technique was a valuable contribution to
this study that helped us to visualize the membrane organization of SCV/SIF. The use of
HPF-FS instead of conventional EM preparation was a key step for fine ultrastructural
preservation of intact membranes of SIF, SCV and Salmonella. The generation of tomograms
and their reconstruction then further helped us to understand the 3D complexity of SIF/SCV,
namely the spatial arrangement of membranes which is otherwise difficult to interpret only
from 2D projections of thin sections. Thus we provided the first high-resolution 3D image of
Salmonella contained within the SCV of an infected cell. The remarkable and novel
contribution here with an importance for understanding of SIF biogenesis was visualization of
the SCV as a complex of interconnected multi-membrane compartments where membrane can
be extended and folded to form an open hollow membrane tubes that contain cytoplasm.
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Materials and Methods
Bacterial strains and culture conditions
Salmonella enterica serovar Typhimurium NCTC12023 was used as the wild-type
strain and all mutant strains were isogenic to this strain. For certain experiments, mutant
alleles were transduced into strain background LT2A. We have previously compared
intracellular phenotypes of wild-type strains NCTC12023 and LT2A and did not observe
differences in the induction of SIF (Rajashekar et al., 2008).
Cell culture
Human epithelial cell line HeLa cells (ATCC No CCL-2) were cultured in DMEM
with 10 % FCS, penicillin and streptomycin, and grown in 37°C in a humidified atmosphere
containing 5 % CO2. Another cell line used in this study was murine macrophage like cell line
RAW 264.7 (ATCC No. TIB-71), which was also grown as described above. For infection of
epithelial cells, all the strains were grown overnight in LB broth with carbenicillin at
100g/ml and sub-cultured for 3.5 h until the cultures reach late log phase and then used for
infection at MOI of 50. For infection of RAW, firstly cells were stimulated with 5ng/ml IFN
for 16 to 18 h, which resulted in about 90 % activated macrophages as judged from the cell
morphology. The bacterial strains were grown overnight until stationary phase was reached
and then directly used for infection at an MOI of 50. HeLa cells were transfected with 500g
of plasmid DNA (LAMP-1-GFP kindly provided by Patrice Boquet) were mixed with the
transfection reagent and added to 3mm disc where cells were grown. Cells were incubated for
4 to 5 h, then medium was changed and fresh DMEM with 10 % FCS was added. Cells were
used for infection studies 16 to 18 h after transfection.
Preloading of BSA-gold
HeLa cells were grown on UV-sterilized and poly-L-lysine-coated sapphire discs (3
mm) in glass-bottom dishes (MatTek Corp). To label the endocytic pathway of infected cells
from early endosomes to lysosomal compartments the cells were pulsed with 10nm BSA-gold
(final concentration of OD520=5) for 6 h (3-9 h post infection), washed and chased for 1h
before processing for cryo-fixation. BSA-gold was prepared as described earlier (Slot &
Geuze, 1985).
Sample vitrification and freeze substitution (HPF-FS)
At the indicated time points usually 10 h post infection, the sapphire discs with cells
were removed from dishes, manually transferred to the HPF device (HPM010, Baltec) and
processed by cryo-fixation within about 10 sec.
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Briefly: Sapphire discs were put onto the flat surface of 3mm aluminum carrier (Type B,
Baltec) in the open cleft of the HPM-010 holder and covered with another carrier (Type A,
Baltec) with a central cavity of 2mm in diameter and 100 to 200µm in depth). Both carriers
were immersed in Hexadecen prior use but no additional cryo-protectant was used in this
setup. The excess of medium and hexadecen was blotted out by filter paper and holder was
immediately inserted to the HPM-010 for cryo-fixation processing. After freezing the samples
were transferred in cryovials into the AFS-1 device (Leica) under liquid nitrogen and
processed for freeze substitution and plastic embedding as follows: incubation in 1% OsO4 +
0.1% Uranyl Acetate in Aceton (40 h at -90°C); warm up to -30°C (12 h, 5° per hour); 3 h
incubation at -30°C; warm up to 0°C (6 h, 5° per hour); wash in acetone and infiltration with
increasing concentrations of EPON812 (Serva) (1:3, 1:1 and 3:1 in acetone, 2h each);
infiltration in pure EPON (O/N at RT). Samples were finally transferred into the PP cylinder
holder (Leica) for flat-embedding, infiltrated in fresh EPON (4h, RT) and polymerized for 48
h at 60°C.
EM and ET
Sapphire discs were removed from the blocks face by liquid nitrogen and thin (40-
60nm) or thick sections (250-350nm) parallel through the cell monolayer (xyz-sections) were
cut on Leica ultra-microtome (UCT) with a diamond knife. Sections were post-stained with
lead citrate (2% in water) and examined on 120kV electron microscope Biotwin CM120
Philips equipped with a bottom-mounted 1K CCD Camera (Keen View, SIS). Manual
tracking and drawing in Photoshop (Adobe) was used to create a mask layer for visualization
of SCV/SIF membrane contours in the EM micrographs of thin cross-sections.
The thick sections (300nm) of HPF/FS samples were placed on a slot grid covered
with a formvar film and decorated with fiducial gold particles (10nm protein-gold) on both
sides for image alignment. Grids were placed in a high-tilt holder (Fischione Model 2020) and
single or dual-axis ET was carried out using a Tecnai F30 (FEI) electron microscope
(operated at 300 kV) equipped with a field emission gun and a 4084 x 4084 pixels CCD
camera (Eagle, FEI). Digital images (bin 2, 2048 x 2048) were taken every 1°, over a ± 60°-
65° range with a pixel size ranging from 0.5-1 nm using Serial EM acquisition software with
robust prediction of specimen movement (Mastronarde, Boulder, Colorado).
IMOD software was used for alignment; 3D reconstruction and merging of the serial
tomograms and the volume segmentations were performed with the Amira 4.1 Visualization
package (Visage Imaging, Berlin, Germany).
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4 DICUSSION
In this study, the intracellular activities of Salmonella have been described in real time
using live cell imaging as well as by ultrastructural analyses. Salmonella is an intracellular
pathogen and once inside the host it establishes a niche for its survival using its virulence
traits (reviews in Haraga et al., 2008). The main factors which determine the virulence of
Salmonella is the secretion of host of effector proteins that are encoded on SPI1 and SPI2
Type III secretion systems (see section 1.4). These effector proteins help the bacteria to invade
the host cells and cause intracellular replication and thus ensure spread of bacteria and results
in pathogenesis and clinical conditions (typhoid or gastroenteritis).
The successful outcome of Salmonella infections depends on how the pathogen
utilizes the host components for its survival. Therefore, Salmonella has to create an
environment in the intracellular life where its virulence factors have efficiently made use of
host machinery like the vesicle trafficking (endocytosis and exocytosis), cytoskeleton
(microtubule and actin filaments), motor proteins, etc. to be a successful pathogen.
Intracellular phenotypes of Salmonella
Comparison of general tubular structures and SIF
One of the common phenomena in mammalian cells is the formation of tubular
organelles, for transport compartments from Golgi to plasma membrane (Bonifacino et al.,
2006) or sorting endosomes (Driskell et al., 2007). For example (Vyas et al., 2007) authors
(Fig 10) observed tubular structures with a high content of major histocompatibility complex
(MHC) II in DC. Such tubules are formed without the involvement of intracellular bacteria.
The common feature of Salmonella infection is also to form tubular aggregates of endosome
called SIF for ‘Salmonella-induced filaments’. The comparison of cells infected with WT and
SPI2 Salmonella or mock-infected cells revealed the morphological difference between SIF
induced by Salmonella and the intrinsic tubular comportments. The tubular endosomal
structures observed in Salmonella WT-infected HeLa cells had dynamic properties and due to
their length were clearly distinguishable from shorter tubular endosomes that appeared in non-
infected HeLa or cells infected with a SPI2-deficient strain (Fig 5B from Chapter 1).
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Fig 10: Comparison of normal tubular structure and SIF in BM-DC. A) Normal tubular structures found on BM-
DC (adopted from Vyas et al., 2007) (left panel). B) SIF in WT Salmonella infected BM-DC pulsed with
Alexa568-dextran (right panel). Scale bar, 5m
SIF in various cell types
The formation of SIF were initially restricted to epithelial cells (Garcia-del Portillo et
al., 1993), but our observations indicate that dynamic SIF were present in macrophages and
BM-DCs (Rajashekar et al., 2008) as well which is independent of bacterial invasion and
therefore it could be concluded that SIF have general properties across the cell types tested.
Initially, SIF formation was not observed in macrophages, probably due to the small size and
spherical form of the cells. However, RAW macrophages as well as primary macrophages
were used in this study to monitor SIF,. We activated macrophages with IFN, resulting in
morphological changes of the cells, i.e. the formation of adherent and flattened cell with
highly phagocytic properties. Due to this, macrophages are highly activated and we could see
onset of SIF at a late time point post infection (8 h p.i). This was also the same situation with
BM-DC and with very less significant replication which has been previously shown (Jantsch
et al., 2003). However a low number of bacteria in DC were sufficient to induce formation of
dynamic SIF. Therefore replication of intracellular bacteria can not be linked to SIF formation
as previously proposed (Birmingham et al., 2005). SIF induction and dynamic properties of
SIF were observed after uptake of non-invasive WT bacteria or SPI-T3SS mutant, the
phenotypes described here are therefore independent of SPI1 function.
Normal BM-DC Salmonella infected BM-DC
SIF Normal tubular endosomes
A B
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Properties of SIF formation and dynamics
HeLa epithelial cells were used as the model cell culture throughout this study to
investigate the properties of SIF formation and dynamics. A detailed kinetics was performed
to study the onset of SIF and progression through the course of infection. SIF were formed as
early as 3h post infection and we also observed a dramatic remodeling of the host endosomal
system. Early after the onset of SIF formation (3.5 - 5h p.i.), the velocity and the variability
in SIF appearance was most prominent, while at later points (> 7 h p.i), a complex network of
SIF was established that exhibited only little extension or contraction. This could be explained
due to the continuous integration of the endosomal membranes into SIF, resulting in a
shortage of available membranes at later time points of infection. Also one of the striking
properties of the SIF was that it randomly extended, retracted, branched, and also the long
tubular structures broke and joined other SIF network (Rajashekar et al., 2008). This later
paved way for a static SIF network that hardly showed growth or retraction. We quantified the
dynamics of SIF by kymographs which are time space-plots. However, we observed from
these results that SIF did not grow with the same velocity as that of MT and but SIF followed
the growing MT tracks for its establishment.
SIF dynamics and microtubules
It had been also previously shown that SIF formation requires the host cell
cytoskeleton namely microtubules for it formation (Garcia-del Portillo et al., 1993).). It has
also been shown that intracellular Salmonella can induce, in a SPI2-T3SS dependent manner,
the bundling of microtubules of infected host cells (Kuhle et al., 2004). There is a clear role of
MT-based motility, as indicated by the effect of nocodazole or taxol on SIF dynamics and the
function of dynein and kinesin motors has been reported (Guignot et al., 2004; Harrison et
al., 2004). However the specific contribution of individual motor proteins in the dynamics of
SIF extension is not known till date. Experiments involving depolymerization of MT by
pharmacological agents such as nocodazole arrested the dynamics of SIF at early time points.
However, at late time point when SIF had established a static network, there was no effect of
nocodazole treatment (Fig 8B from Chap-1). This observation might indicate structural
differences between 'early' dynamic SIF and 'late' non-dynamic SIF. Our ultrastructural
analyses showed that SIF can form along MT and that SIF can be attached to two or more
microtubules (Fig 8A from Chap-1). The cross-linking activity might be mediated by effector
proteins that are present in the membranes of SIF and bind directly to microtubules or
indirectly via microtubule-associated proteins.
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Molecular basis for SIF dynamics
We have proposed a model (Fig 9, Chap-1) in order to explain the dynamics of SIF at
the cellular level. SIF growth can be directed towards –ends or +ends of MT, depending on
the proportion of dynein or kinesin motors, respectively, that are recruited. SIF extension and
contraction can be explained by the fact that every fusion step will introduce membrane
material into the SIF and partially and temporarily relax the internal stress. However, when
the membrane tip of SIF is pulled by MT motors too far without fusion, the elastic stress in
the membrane increases and reaches a critical threshold. This might lead to the detachment of
motors from MT or from SIF membrane; where in the SIF collapses rapidly relaxing
membrane elastic stress (Fig 11).
Fig 11: Cartoon showing the model for SIF extension and collapse based on movement of molecular motors like
kinesin and dynein. A) SIF extending from SCV due to pulling force of kinesin motor protein. B) Retraction of
SIF due to high pulling force of motors which looses contact with either SIF or the microtubule track. (Adopted
from Rajashekar et al., 2008)
The higher velocity of SIF contraction compared to extension would be in line with
such model. Although SIF extension in both direction can be observed, extension towards the
+ends dominates over the time course of infection indicating a preferential recruitment of
kinesin or cargo transported by kinesin. The appearance of a stabilized SIF network in later
phase might indicate a consumption of vesicles available for fusion. During the early phase of
intracellular life of Salmonella, SIF grow out from the SCV by both continual fusion of
A
B
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vesicles with the tip of SIF and by pulling force generated by MT motors associated with the
tips of SIF.
Redirecting of endocytic and exocytic traffic by SIF and SISTs (Salmonella-
induced SCAMP3 tubules)
The protection of intracellular Salmonella against antimicrobial effectors is only one
of the many consequences of the interference with host cell transport processes. SIF are
LAMP1-positive and have a membrane composition similar to that of the SCV (Garcia-del
Portillo et al., 1993). SIF formation requires microtubules, but not the actin cytoskeleton,
although these filaments can be decorated with actin (Brumell et al., 2002; Kuhle et al., 2004).
Possible mechanisms that contribute to SIF formation include repetitive initiation of vesicular
budding from the SCV, in which fission events are incomplete, or continuous fusion of
endocytic vesicles with the SCV, which would result in endosomal tubulation or elongation of
the SCV.
Fig 12: This is a still image from a movie showing SIF and SISTs. HeLa cells were transiently transfected with
pEGFP-SCAMP3 and with pLAMP1-HcRed and infected with WT S. typhimurium expressing DsRed. The
lower panel is a 6x magnification of the inset. The red arrowhead indicates a SIF having nearly undetectable
EGFP-SCAMP3; the green arrowhead indicates a SCAMP3 tubule with no detectable LAMP1-HcRed (a SIST);
and the yellow arrowhead indicates a SIF containing SCAMP3. All Scale bars, 5 m. Adopted from (Mota et al.,
2009).
Studies on the fate of exocytic markers in Salmonella-infected cells indicated that the
normal route of exocytic transport was also affected. A redirection of Golgi transport to the
SCV instead of transport to the plasma membrane was observed (Kuhle et al., 2006). More
recently, the formation of a second tubular network was observed that is distinct from the SIF
network. The second network is characterized by membrane proteins of the trans-Golgi
network (TGN) such as SCAMP3, indicating the remodeling of exocytic transport (Mota et
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al., 2009). Formation of this network is induced by Salmonella WT, and the network is termed
'Salmonella-induced SCAMP-3 tubules' (SIST) (Fig 12). SIST and SIF partially overlap and
tubular membranes with endosomal and TGN markers were observed. In addition, tubular
structures with endosomal markers lacking TGN markers, and vice versa, were found (Mota
et al., 2009). These researchers also found that the formation of both types of tubular
networks depends on the function of the SPI2-T3SS since neither SIST nor SIF were induced
in cells infected with a SPI2-T3SS-deficient strain. These observations demonstrate that as a
consequence of the modification of host cell transport, aggregation of vesicles in both the
endocytic and the exocytic pathway occurs.
The particular appearance of tubular aggregates can be explained by the involvement
of microtubules as transport routes for vesicular traffic. In the case of SIF induction, a specific
involvement of microtubule motor proteins was observed, and a subset of the SPI2-T3SS
effector proteins required for SIF inductions interferes with motor proteins such as kinesin or
dynein or their adaptors or effectors (reviewed in Abrahams & Hensel, 2006; Barkowski et al.,
2008).
Contribution of effector proteins to SIF formation and SCV positioning
The function of the SPI2-T3SS has been linked to various intracellular events such as
the avoidance of antimicrobial activities, modification of the MT and actin cytoskeleton,
interference with antigen presentation and many others (reviewed in Haraga et al., 2008).
SPI2 effectors phenotypes have been shown for SifA, SseF, SseG, PipB2, SopD2, SpiC, etc.
(Guy et al., 2000; Kuhle & Hensel, 2002; Jiang et al., 2004, Knodler & Steele-Mortimer,
2005; Boucrot et al., 2005; Beuzon et al., 2000; Brumell et al., 2001; Stein et al., 1996). We
screened all the SPI2 secreted effectors for its contribution to SIF formation by generating
mutants lacking a particular effector under study. By infecting deletion mutants lacking
effector to HeLa cells we found two distinct types of phenotypes exhibited by sseF and pipB2
deletion mutants. In sseF deletion mutant due to the absence of SseF protein we could see that
SIF are still formed but are morphologically thinner that the WT SIF (as verified by
intensities) (Fig 13). This also disproves the earlier notion (Kuhle et al., 2002) that lack of
SseF causes “Pseudo-SIF”. We found that the occurrence of Pseudo-SIF were a consequence
of fixation as these structures are unstable to fixatives and thus was considered as fixative
artifacts. On the other hand, a further novel observation revealed by the live cell analyses was
the unique structure of endosomal aggregates observed in a fraction of the cells infected with
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the pipB2 mutant strain. PipB2 is previously shown required for the reorganization of LE/Lys
define during Salmonella infection (Knodler et al., 2008).
Our study shows that a subset of cells infected with the pipB2-deficient strain showed
the aggregation of LAMP-1-GFP positive vesicles which we term “bulky SIF” (Fig 4 for
comparison) It could thus be proposed that the occurrence of these structures may be due to
lack of PipB2 to engage MT plus end kinesin motors for extension of LAMP-1-positive
tubules from cell centre to cell periphery. The most important effector of the SPI2-T3SS is
SifA. It plays a central role in the intracellular replication of Salmonella (Beuzon et al., 2000).
Also mutants lacking sifA loose the membrane of the SCV and are largely found in the
cytoplasm as seen in live time lapse movie and in electron micrographs. This is in accordance
with the previous reports (Brumell et al., 2002).
A role of SPI2-T3SS effector proteins in controlling the MT motor protein activities
acting on the SCV has been reported (Boucrot et al., 2005; Henry et al., 2006). According to
current models for the function of SPI2-T3SS effector proteins, the SCV has to maintain a
balance between opposing activities of motor proteins (Abrahams et al., 2006).
Fig 13: HeLa cells Infected with WT, sseF and pipB2 mutants respectively. It shows the comparison between SIF
induced by the deletion mutants sseF thin SIF (see inset) and pipB2 bulky SIF (see inset) as compared to WT.
Scale bar, 5m
Such a balance would allow the proper intracellular positioning of the SCV and the
sufficient supply of endosomal membranes to maintain the SCV with an increasing bacterial
population. These models might have to be reconsidered in the light of our observations of the
Bulky SIF Thin SIF Normal SIF
WT sseF mutant pipB2 mutant
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highly dynamic nature of SIF. For example, the proposed function of SifA as an effector that
interacts with SKIP in order to prevent the kinesin motor protein activity on the SCV might
not be sufficient to explain the induction of SCV tubulation and the rapid extension or
contraction of SIF.
SIF ultra-structure and model for SIF biogenesis
This study provides the first evidence of the ultra-structure of SIF and its biogenesis and
based on our observations using electron tomography. This study provided an insight into
membrane organization with respect to SIF and SCV. We were able to detect SIF that we
describe as long tube originating from the SCV filled with lysosomal or cytoplasmic contents.
The average diameter of SIF is in the range of 160 nm (± 39 nm).We could also show that SIF
are aligned on the MT providing more detailed evidence that SIF require MT as scaffold for
their formation (Fig 8A, Chap-1).
Morphologically variant SIF induced by sseF and pipB2 mutant strains could be further
reconfirmed though EM at the structure level. It was quite convincingly shown that thinner
SIF induced by sseF mutant and bulky SIF induced by pipB2 mutant was traced by EM (Fig 7
chap-2). This added to the confidence to our microscopy results that were of limited
resolution.
In addition to this we could show SIF with two types of membrane structures. First type
was SIF with double delimiting membranes with cytoplasmic content, and the second type
showed single delimiting membranes with lysosomal content (Fig 1 Chap-3). We further
deciphered the nature of these two subpopulations of SIF and tried to generate a model for the
biogenesis of SIF during the course of infection (Fig 14C). One of characteristic observations
from the light microscopy was the occurrence of “tracking SIF” and “trailing SIF” (Fig 14A).
Here less intense thinner SIF or “tracking SIF” were growing with faster pace and were
followed by thicker “trailing SIF”. Our model is based on this concept and we have proposed
that the double membrane SIF emerged from the single membrane SIF which can be
explained by “spontaneous membrane remodeling”. Here we suggest that this interchangeable
ultrastructural characteristic of SIF is a result of spatio-temporal remodeling (Fig 14B) of the
excess membrane (folding, extension, wrapping etc.) that cumulates on the SCV as a result of
continuous fusion with the endocytic vesicles. From the mechanistic view, all membrane
fusion/fission processes within cells result in dis-balance of surface to volume ratio and/or in
changes of membrane surface tension on the resulting organelle or membrane vesicle.
Modulation of transient changes in membrane properties is tightly dependent on protein and
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lipid composition and their spatio-temporal distribution, but how these processes are regulated
in cells is still poorly understood.
A cartoon in Fig 14C shows the overall model for SIF formation and biogenesis. Here
the cartoon explains the initial dynamic SIF formed early on post infection and the complex
network SIF formed towards the late time points post infection. The biogenesis of SIF
between these two stages is a complex event which involves reorganization of membranes in
the SIF and SCV. The cartoon also illustrates the “tracking SIF” and the “trailing SIF” and the
structural difference between them as shown in longitudinal and cross sections. The tracking
SIF are the growing SIF which encompass more and more late endosomal and lysosomal
contents and is of single membrane structure. The trailing SIF follows the tracking SIF
enwrapping the cytoplasmic contents and are of double membrane nature due to membrane
invagination.
Biological role of SIF
Nutritional adaptation to intracellular life
The biological role of the induction of SIF and the remodeling of the endosomal
compartment of the host cell of Salmonella is not completely understood. It has been
previously proposed that SIF formation is required for the replication of intracellular
Salmonella (Garcia-del Portillo et al., 1993). However, the formation of SIF in cell types like
BM-DC where Salmonella per se does not replicate shows that SIF may be required for other
functions rather than replication. It has also been recently published that SCV is continuously
interacting with the endocytic pathway (Drecktrah et al., 2007.). This also led us to perform
some fluid tracer assay where we applied various methods for the labeling of the luminal
content of SIF as well as the membranes and both approaches resulted in the appearance of
SIF with similar morphology and dynamic properties.
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Fig 14: Model for existence of single membrane and double membrane SIF: A) Video time lapse movie series of
HeLa cells transfected and infected with WT Salmonella showing “tracking” and “trailing” SIF. B) Cartoon
showing the “Membrane Organization Process” where Single membrane SIF acquires more and more membrane
material, folds and in the process enwraps the cytoplasmic bubble and reorganizes to form double membrane
SIF. C) Cartoon showing the events involved in the biogenesis of SIF. Scale bar, 5m
Longitudinal section
Longitudinal section
SIF formation during the course of infection
SIF
Initial dynamic SIF Non-dynamic network
Lagging SIF
Double delimiting membrane Single delimiting membrane
Leading SIF
SIF Biogenesis
Cross section Cross section
Late endosomes
Salmonella
Cytoplasmic contents
Microtubule
Actin
SCV
Salmonella Invasion
C
Tracking SIF Trailing SIF Double membrane
SIF or Trailing SIF
Single membrane SIF or
Tracking SIF
A B
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Interestingly, we could observe that the lumen of SIF as well as of the SCV was
accessible to fluid phase markers in various Salmonella-infected host cells. These data
supported the model of (Drecktrah et al., 2007) and stand against the currently prevailing
model that the SCV is separated from the endosomal system. This led us to propose or
speculate that SIF are necessary to provide nutrition supply to the metabolically active
Salmonella in the SCV.
In contrast to bacteria that are highly adapted to life inside cells, most serotypes of S.
enterica are fully prototrophic bacteria that can use a variety of carbon compounds for
biosynthesis of macromolecules and as sources for energy. Various researchers observed a
rapid intracellular growth rate of Salmonella within host cells and the generation time of
intracellular Salmonella may be similar to that of bacteria growing in rich media without
limitations for nutrients or trace elements. This observation indicates that intracellular bacteria
have sufficient access to nutrients to allow rapid replication. The actual source of nutrients
within eukaryotic host cells is still a matter of debate. Assuming that the SCV is a
nutritionally poor environment, the bacteria may adjust their metabolism to the utilization of
those compounds that are in sufficient amounts present in the SCV. For example, using a
reporter strain of S. enterica serovar Typhimurium or microarray analyses, it has been
observed that high affinity uptake systems of Mg2+ and iron are induced in intracellular
bacteria (Eriksson et al., 2003; Garcia-del Portillo et al., 1992). Data for similar analyses of
the carbon metabolisms are not available. We have investigated the role of various metabolic
pathways in the intracellular replication of S. enterica serovar Typhimurium within
macrophages (unpublished observations). These analyses indicated that none of the individual
mutations or combinations of mutations caused a reduction in intracellular replication that
was as strong as that of SPI2-T3SS deficient strains.
An alternative model for the source of intracellular nutrition of Salmonella results from
gross remodeling of the vesicular trafficking of the host cell. As stated above the fluid tracers
are seen in SCV as seen in the light microscope, however this is not strong evidence as there
is limited resolution to light microscopes. Therefore we tried a similar experiment using BSA-
Gold nanoparticles and performed EM/ET to see if these particles end up in the lumen of the
SCV. Though our results convincingly showed that BSA-Gold nanoparticles are present in the
internal lumen of the SCV, we can only speculate that continual fusion of SCV with
endosomes brings nutrients for replicating Salmonella within SCV, but not the SIF. If SIF are
truly the “food pipes” for the SCV has yet to be proven experimentally.
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It will be an important challenge for future research to determine whether these nutrient are
host-derived macromolecules or metabolites directly utilized by Salmonella, or if the bacteria
directs host cell vesicular transport in order to 'hijack' vesicles containing internalized
nutrient.
Conclusions and open questions
In light of the new observations described above, current models for the biogenesis of
SIF have to be revised. The SCV is a highly dynamic compartment and by means of virulence
factors such as the effectors of the SPI2-T3SS, intracellular Salmonella control the positioning
of this compartment as well as fusion events with endosomal and exocytic vesicles. The
pathogen also transforms the entire endosomal-membrane system of the host cell. The recent
observation of SIST as a second tubular membrane system raises the question if even more
tubular membrane structures exist and further membrane markers should be investigated. The
dynamics and complexity of the resulting unique membrane structures will require further
detailed studies on the biogenesis. In continuation of the previous studies that led to new
insights into the intracellular lifestyle of Salmonella, both EM with high spatial resolution and
live cell imaging with high temporal resolution will play important roles in this process.
Although ultrastructural analyses could lead to novel insights into the intracellular
biology of Salmonella, there are currently two major limitations, i.e. the highly dynamic
characteristics and the heterogeneity of the intracellular phenotypes. An important approach to
overcome these limitations will be the correlative EM technique, which will allow addressing
the ultra-structure of individual intracellular bacteria, SCV or SIF directly after observation in
a live cell setup. Another limitation of ultrastructural investigations, i.e. the restriction to
analysis of a rather small volume of the cell, might be circumvented by techniques such as
EM tomography that might allow the three-dimensional reconstruction of novel membrane
compartments with ultimate resolution. Yet, the next challenges will be the molecular
understanding of the host factors that contribute to Salmonella infection. We still have certain
open questions that have to be addressed like:
a) Is the SCV a segregated compartment or does it interacts with vesicular system in host
cells? If so, how does it protect itself from harsh intracellular environment which has
antimicrobial agents and nutritionally stringent condition?
b) What is the biological function of SIF?
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c) Are SIF confined to only cell culture models or does it also occurs in-vivo under natural
conditions in cell of the gut? If so, how could this contribute to final outcome of disease?
We envision that combinations of such state of the art approaches of imaging will
unveil further more secrets of the intracellular life of Salmonella.
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5 SUMMARY
Salmonella is a facultative intracellular pathogen with a broad range of hosts and
causes different outcome in various hosts. The majority of virulence factors of Salmonella are
encoded by genes that lie in its pathogenecity islands which are classified as SPI1 and SPI2.
The SPI and SPI2 each encode type three secretion systems and set of effector proteins that
are responsible for host cell invasion and intracellular pathogenesis respectively. Among
various intracellular phenotypes of Salmonella that have been reported, Salmonella-induced
filaments (SIFs) is one of the most important phenotypes of Salmonella infection in cell
culture models. Inside a cell, Salmonella resides in a special compartment called as
Salmonella containing vacuole (SCV). Later in the infection stage, SCV extends to SIFs, a
phenotype described using fixed cells and thought to arise as a result of continuous fusion of
endosomal vesicles, rich in lysosomal glycoproteins and were previously described in fixed
cells. Further, SIFs are highly dynamic structures and actively involve host trafficking system
such as the endo and exocytic pathways. In the present investigation, we wanted to establish
live cell assays to study in more detail the formation of SIF, its progression through the course
of infection, and more precisely analyze the dynamics of SIF growth, retraction and
contribution of the host cytoskeletal elements in the formation of these novel structures.
Using live cell setup, we found that SIFs were formed as early as 3 h post infection
and were highly dynamic structures. Based on our results, we propose that the formation and
growth of SIFs is a result of continuous fusion of late endosomes and lysosome membranes
with the SCV. Initially between 4 to 6 h post infection, SIFs were found to be highly dynamic
with rapid extension and retraction. However, during later stages of infection they established
a complex SIF network which was static with rare extensions or retractions. Our results also
revealed that both the SIF formation and dynamics was dependent on microtubules. SIFs are
not only confined to HeLa cells but are also observed in other cell types such as macrophages
and BM-DC highlighting that SIF formation is a more general phenomenon that mainly
remodel the host endosomal system enabling bacterial survival. The fluid phase pulse chase
studies showed a constant interaction between SCV and the endocytic pathway and thus
negated the previous belief that SCV was a separate compartment and did not fuse with late
endosomes and lysosomes. Similar to previous reports, screening for various SPI2 effectors
for their contribution to SIF formation identified that the absence of effector protein SifA
completely abolished the occurrence of SIFs and together with clear loss of SCV around the
Salmonella (Beuzon et al., 2000).
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In addition to the loss of SIF formation, the screening also revealed novel SIF
phenotypes. While the loss of effector SseF, induced SIFs that were morphologically thinner,
Salmonella mutant deficient in effector PipB2 lead to SIFs that were non-dynamic, bulky
aggregates of late endosomes called as “Bulky SIFs”. Thus SifA, SseF and PipB2 played an
important role in SIF formation and dynamics. Finally EM studies reconfirmed our live cell
assay results like SIF formation on MT, SseF and PipB2 phenotypes of “thin and bulky SIFs”.
The striking revelation came from electron microscopy and tomography analysis
which lead to certain novel observations on SIF biogenesis and SCV membrane organization.
From these studies we came to the conclusion that SCV and SIF undergoes “spontaneous
membrane re-organization” with initial formation of single membrane de-limiting SIFs and
further by encompassing more and more membrane and cytoplasmic material forms SIFs that
have double de-limiting membranes. A 3D model generated gave a clear picture and
interpretation of how such membrane reorganization takes place. Based on these results, for
the first time we have proposed a model for the biogenesis of SIFs and SCV at the
ultrastructural level.
This work also presents Salmonella as a perspective model for study of interactions
between the pathogen and the host cell that drive complex membrane remodeling and high-
jacking of endosome trafficking to favor growth and survival of the pathogen within the host.
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Zusammenfassung
Salmonella ist ein fakultativ intrazellulärer Krankheitserreger mit einem breiten
Wirtsspektrum und verursacht Krankheitsbilder mit unterschiedlichen Folgen in
verschiedenen Wirten. Die meisten Virulenzfaktoren von Salmonella sind von Genen kodiert,
die auf Pathogenitätsinseln liegen, die als SPI1 und SPI2 bezeichnet werden. SPI1 und SPI2
kodieren jeweils Typ-III-Sekretionssysteme und einige Effektorproteine, welche für die
Invasion bzw. intrazelluläre Pathogenese verantwortlich sind. Unter den verschiedenen
intrazellulären Phänotypen von Salmonella, sind Salmonella-induzierte Filamente (SIFs) einer
der besten und meist studierten Phänotypen der Salmonella Infektionen in
Zellkulturmodellen. Salmonellen befinden sich in besonderen Vakuolen, die als Salmonella
containing vacuoles (SCV) bezeichnet werden und welche später SIFs ausbilden. SIFs sind
endosomale Vesikel, die reich an lysosomalen Glykoproteinen sind und wurden kürzlich in
fixierten Zellen beschrieben. SIFs sind hoch dynamische Strukturen und beanspruchen die
Transportwege des Wirtes, wie die endo- und exozytotischen Wege. In dieser Arbeit wollten
wir Lebendzell-Experimente etablieren um die Bildung der SIFs und deren Veränderungen im
Laufe der Infektion genauer zu untersuchen. Außerdem wollten wir die Dynamik der
Ausbreitung und Kontraktion von SIFs analysieren und wie das Zytoskelett der Wirtszelle zur
Bildung dieser Strukturen beiträgt.
Wir konnten herausfinden, dass SIFs hoch dynamische Strukturen sind, die schon 3
Stunden nach der Infektion gebildet werden. Aufgrund unserer Ergebnisse vermuteten wir,
dass die Bildung und das Wachstum von SIFs ein Resultat einer ständigen Fusion von späten
Endosomen und lysosomalen Membranen mit der SCV ist. Zwischen 4 und 6 Stunden nach
der Infektion wurden die SIFs als hoch dynamische Strukturen gefunden, die sich schnell
ausbreiteten und kontrahierten, während sich zu späteren Zeitpunkten ein komplexes SIF
Netzwerk gebildet hatte, welches statisch war und kaum noch Ausbreitung oder Kontraktion
zeigte. Außerdem konnten wir zeigen, dass die SIF-Bildung und Dynamik von Mikrotubuli
abhängt. SIFs sind aber nicht nur auf HeLa Zellen beschränkt, sie konnten auch in anderen
Zellsystemen wie Makrophagen oder BM-DC gefunden werden, was zeigt, dass die SIF
Bildung ein eher allgemeingültiges Phänomen ist, welches durch die Nutzung des
endosomalen Systems des Wirtes das bakterielle Überleben ermöglicht. Pulse chase
Experimente zeigten, dass SCVs in ständiger Interaktion mit dem endozytotischen Weg sind,
und somit konnte der bislang existierende Glaube entkräftet werden, dass die SCV ein
separates Kompartiment ist, welches nicht mit späten Endosomen und Lysosomen fusioniert.
Im Einklang mit früheren Studien konnten wir bei der Suche nach SPI2 Effektoren, die zur
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SIF Bildung beitragen, feststellen, dass eine SifA Mutante das Auftreten von SIFs komplett
verhindert und ebenso konnte das Fehlen einer SCV festgestellt werden (Beuzon et al. 2000).
Zusätzlich zum Verlust der SIF Bildung, brachten unsere Studien auch neue SIF Phänotypen
hervor. Während der Verlust des Effektors SseF SIFs induzierte, die morphologisch dünner
waren, führten Mutationen in PipB2 zu statischen SIFs, die aus klumpigen Aggregaten von
späten Endosomen bestanden, diese wurden deshalb als „bulky SIFs“ bezeichnet. Hiermit
konnte gezeigt werden, dass SifA, SseF und PipB2 eine wichtige Rolle in der SIF Bildung
und Dynamik spielen. Zuletzt konnten auch elektonenmikroskopische Studien die Ergebnisse
der Lebendzell-Experimente bestätigen.
Die bemerkenswerteste Beobachtung kam jedoch durch unsere elektronen-
mikroskopischen und tomographischen Experimente auf, welche zu einigen neuen
Beobachtungen in der SIF Biogenese und SCV Membranorganisation führten. Durch diese
Studien kamen wir zu dem Entschluss, dass sich SCV und SIF spontanen
Membranumlagerungen unterziehen, die mit der Bildung von Einzelmembranen beginnt, um
dann weiteres Membran- und zytoplasmatisches Material zu akquirieren wodurch dann SIFs
mit einer Doppelmembran gebildet werden. Ein 3D-Modell zeigte ein klares Bild wie solche
Membranumlagerungen stattfinden können. Durch diese Ergebnisse konnten wir zum ersten
Mal ein Modell für die Biogenese von SIFs und SCV auf ultrastrukturellem Niveau darstellen.
Diese Arbeit stellt Salmonella als aussichtsreiches Modellsystem dar, um Wirts-
Pathogen Interaktionen zu untersuchen, welche komplexe Membranumlagerungen und
Ausbeutung des endosomalen Netzwerkes involvieren und dadurch das Überleben und das
Wachstum des Pathogens im Wirt sicherstellen.
133
6 LIST OF PUBLICATIONS AND CONTRIBUTION OF CO-AUTHORS
1. Dynamic remodelling of the endosomal system during formation of Salmonella induced
filaments by intracellular Salmonella enterica.
Rajashekar R, Liebl D, Seitz A, Hensel M.
Traffic. 2008 Dec; 9(12):2100-16. Epub 2008 Sep 19.
PMID: 18817527 [PubMed - indexed for MEDLINE]
Other manuscripts under preparation
2. Novel functions of SPI2 effectors during intracellular pathogenesis of Salmonella enterica
revealed by live cell and ultra structural analyses.
Roopa Rajashekar, David Liebl, Deepak Chikkaballi, Micahel Hensel
3. Ultra-structure analysis of SCV and Biogenesis of SIFs by Electron tomography
David Liebl, Roopa Rajashekar, Peter Chalenda, Deepak Chikkaballi, Michael Hensel
4. Book Chapter
Title of Book- Salmonella: From Genome to Function Publisher- Caister Academic Press Editor- Steffen Porwollik Vaccine Research Institute of San Diego, 10835 Road To The Cure, Suite 150, San Diego, CA 92121, USA Contributed to Chapter 11 titled: The intracellular lifestyle of Salmonella enterica and novel approaches to understand the adaptation to life within the Salmonella-containing vacuole.
Roopa Rajashekar and Michael Hensel
Contribution of Co-authors
David Liebl contributed to all the electron microscopy and tomography studies in this project.
Peter Chalanda did all the 3-D rendering and reconstruction of tomograms. Arne Seitz was
involved in generation of kymograph algorithm and it was extensively used in this study to
generate kymographs for determining SIF dynamics. Deepak Chikkaballi contributed to
scoring of SCV and SIFs induced by different mutants using live cell imaging and
immunostaining.
134
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8 ABBREVIATIONS
ATP Adenosine-5'-triphosphate
BM-DC Bone marrow derived dendritic cells
BSA Bovine serum albumin
CaCo human epithelial colorectal adenocarcinoma cells
CCVs Clathrin-coated vesicles
CCD Charged coupled device
CFU Colony forming units
CFP Cyan fluorescent proteins
DC Dendritic cells
DMSO Di-methyl sulphoxide
DMEM Dulbecco's Modified Eagle Medium
DNA De-oxy ribonucleic acid
EEA1 Early endosome antigen 1
EMBL European molecular biology laboratory
ER Endoplasmic reticulum
EM Electron microscopy
FACS Fluorescence-activated cell sorting
FCS Fetal calf serum
GA Gluteraldehyde
GTP Guanosine-5'-triphosphate
GTPases Singular GTPase
GFP Green fluorescent proteins
HeLa Henrietta Lacks (cell line is name of person from whom it was derived)
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
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HPF/FS High pressure freezing and freeze substitution
iNOS Inducible nitric oxide synthase
IFN Interferon gamma
LAMP Lysosome associated membrane protein
LE/Lys Late endosomes/lysosomes
LPS Lipopolysaccharide
M cells Microfold cells
MDCK Madin-Darby Canine Kidney epithelial cell line
MOI Multiplicity of infection
MHC Major Histocompatibility complex
MT Microtubule
MEM Minimal essential medium
NSF N-ethylmaleimide-sensitive factor
NADPH Nicotinamide adenine dinucleotide phosphate-oxidase
OD Optical density
PBS Phosphate buffered solution
PI3 kinase phosphatidylinositol 3-kinases
PIC pathogen-inhabited compartments
P.I Post infection
PFA Para-formaldehyde
Rabs Family of G proteins
RAW Mouse macrophage cell line
RFP Red fluorescent proteins
ROI Reactive oxygen intermediates
RNI Reactive nitrogen intermediates
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SCAMP Secretory carrier membrane proteins
SCV Salmonella-containing vacuole
SEM Scanning electron microscopy
SIF Salmonella-induced filaments
SISTs Salmonella induced SCAMP Tubules
SKIP SifA and kinesin interacting protein
SNAREs Soluble N-ethylmaleimide-sensitive factor-attachment protein receptor
SPI1 Salmonella Pathogenecity Island 1
SPI2 Salmonella Pathogenecity Island 2
T3SS Type three secretion systems
TGN Trans Golgi network
TEM Transmission electron microscopy
WT Wild type
3D Three dimensional
4D Four dimensional
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9 CURRICULUM VITAE
Roopa Rajashekar
Date of Birth: 30th December 1975
Place of Birth: Bangalore, India
Nationality: Indian
Marital Status: Married with 1 child
Infektion biologie abteilung
Friedrich Alexander Universität
Wassertrum strasse 3-5, D-91052
Erlangen, Germany
E-mail: [email protected]
Phone: 0049-9131-8522114 (off)
0049-9131-1239626 (res)
0049-17620752705 (Handy)
Academic Background
June 2005- Till date: PhD (Molecular Cell Biology and Microbiology) at Infektion Biologie
Abteilung in the group of Prof. Dr. Michael Hensel, title of the thesis: “Novel approaches to
understand the intracellular lifestyle of Salmonella enterica by live cell imaging and ultra-
structural studies"
Sep 1998-May 2000 Master of Science (Biotechnology)
Bangalore University, India
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Jul 1996-May 1999 Bachelor of Science (Microbiology, Chemistry and Botany)
Bangalore University, India
Publications (from current PhD work)
1. Dynamic remodelling of the endosomal system during formation of Salmonella induced
filaments by intracellular Salmonella enterica.
Rajashekar R, Liebl D, Seitz A, Hensel M.
Traffic. 2008 Dec; 9(12):2100-16. Epub 2008 Sep 19.
PMID: 18817527 [PubMed - indexed for MEDLINE]
Other manuscripts under preparation
2. Novel functions of SPI2 effectors during intracellular pathogenesis of Salmonella enterica
revealed by live cell and ultra structural analyses.
Roopa Rajashekar, David Liebl, Michael Hensel
3. Ultrastructure of Salmonella induced filaments as revealed by Electron tomography.
David Liebl, Roopa Rajashekar, Peter Chalanda, Deepak Chikkaballi, Michael Hensel
4. Chapter for Book on Salmonella Title of Book- Salmonella: From Genome to Function Publisher- Caister Academic Press Editor- Steffen Porwollik Vaccine Research Institute of San Diego, 10835 Road To The Cure, Suite 150, San Diego, CA 92121, USA Contributed to Chapter 11 titled: The intracellular lifestyle of Salmonella enterica and novel approaches to understand the adaptation to life within the Salmonella-containing vacuole. Roopa Rajashekar and Michael Hensel
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Workshops/Conferences
Oral presentations
1. Rajashekar, R. Redirection of trafficking of MHC Class II surface molecules during the Salmonella enterica infection. Fachgruppentagung Mikrobielle Pathogenität der DGHM, 19-21. Bad Urach, Ulm, Germany, June, 2006.
2. Rajashekar, R. Interference of cellular trafficking by Salmonella.typhimurium. Prof Hensel and Prof. Hart Inter-group collaboration seminar, ETH Zurich, July 2006.
Posters/Abstracts
1. Rajashekar.R, Seitz.A, Hensel M., Live cell analysis of interference of intracellular
Salmonella with vesicular trafficking of the host cell, ELSO, Dresden, Germany,
September, 2007.
2. Rajashekar.R, Hensel M., Pathogenic Salmonella typhimurium interferes with MHC Class II Cellular Trafficking and Modulates Host Immune Response, DGHM, Wuerzburg, Germany, October 2006.
Workshop and collaboration
Workshop on Basic and Advanced light microscopy and Image analysis at EMBL Heidelberg,
in March 2007. Major part of my PhD project work was also made at Advanced light
microscopy facility at EMBL Heidelberg under the guidance of ALMF research scientists,
headed by Dr.Rainer Pepperkok.
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10 ACKNOWLEDGEMENTS
I would like to extend my deepest gratitude to my mentor Prof. Dr. Michael Hensel for
his excellent supervision, moral support, freedom to work and for his excellent ideas. His
systematic organization and well thought planning have influenced my development as a
future scientist.
My sincere thanks to Prof. Dr. Andreas Burkovski, for being the referee of my
doctoral thesis and Prof. Dr. Uwe Sonnewald and Dr. Andreas Brönrke for being in my
doctoral committee. My special thanks also to the former and present heads of the department
Prof. Dr. Martin Röllinghoff and Prof. Dr. Christian Bogdan for their constant support and
stimulating discussions during our seminar sessions. I would also like to extend my gratitude
to Deutsche Forschungsgemeinschaft (DFG) and the Institute für Infectionbiologische
Abteilung, Universitatsklinikum Erlangen for funding my doctoral work.
I would also like to acknowledge Dr. Reiner Pepperkok, Dr. Arne Seitz, Dr. Stephan
Terjung and Dr. Timo Zimmermann for providing me great opportunity to work at the
Advanced Light Microscopy Facility at EMBL, Heidelberg. I have my heartfelt gratitude for
their support in training me to use light microscopes which was a core part of my PhD work.
My sincere gratitude to Dr. Dipshika Chakravortty for introducing me to Prof Michael
Hensel’s group and for her advice and encouragement during my doctoral tenure.
I would like to extend special thanks to my collaborators Dr. David Liebl and Dr.
Peter Chalanda without whom this project would not have been as successful as it stands
today. I would cherish the interactions from the team work involving them and the immense
knowledge I gained from it. My special thanks to my past and present members from Prof.
Hensel’s group, including Serkhan Halichi, Xin Xu, Wael Hegazy, Felipi-Lopeze Alfonso and
Carolin Wagner for their help and who made my stay pleasant and enjoyable. I would like to
specially acknowledge Dr. Deepak Chikkaballi and Yogitha Jayaswamy for all their moral
support and scientific interactions with Deepak while working in this project. I would then
most importantly like to thank my parents Mr. H.V Rajashekar and my beloved mother late
Mrs.Padma Rajashekar, and my parents-in-law Mr. S.M.Vijaya Chandra and Mrs M.Parvathi
for the support and encouragement to pursue my doctorate studies in Germany. The love,
affection and blessings of my beloved grandmother Mrs. Sharadamma Ramakrishna and Mrs.
Jayamma Ramachandra have given me the strength to travel far in this journey. The moral
support from Mr.Kurt Endres, Mrs. Rita Endres and family in Germany is never to be
forgotten for life. I would like to thank friends, relatives (especially my sister Mrs Deepa
Aravind, and my brother Mr.Ravi Rajashekar and Mrs Anita Ravi and Brother-in-law Mr.
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Shreedhar V and Mrs Lakshmi Shreedhar) and others for all their direct or indirect support
during my PhD studies.
Finally I would like to thank my husband, Mr. Shree Harsha V, for his constant
encouragement in difficult times, his love and support without which I would not have been
the successful person today. His words mean the world to me and his words of courage
boosted my self esteem and made me a better person. A special thanks to my dear little
daughter Arya for having brought lot of joy and zeal into my life.