immunity responds to listeria monocytogenes

Qazvin university of

Medical Science, Fall 1392

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Listeria monocytogenes is a Gram-positive pathogenic bacterium that has adapted to various environments, from soils and food products to the intestinal tract and intracellular compartments of diverse animal species and humans.

The aerobic

Non–spore-forming

Catalse Positive

The organisms are motile at room temperature (25 ̊C) but less so at 37 °C

L. monocytogenes exhibits weak β-hemolysis when grown on sheep blood agar plates.

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L. monocytogenes is a facultative intracellular pathogen

that can live both inside and outside its host.

To infect its mammalian host and to cause the most severe

pathologies, L. monocytogenes is able to cross :

The intestinal

Blood-brain and

Maternofetal barriers

Crossing the host barriers involves bacterial invasion and

survival within a large variety of normally nonphagocytic

cells

It is therefore crucial to understand how L. monocytogenes

induces its own uptake by host cells.

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Listeriosis manifests itself through flu-like symptoms

and can lead to

diarrhea,

meningitis,

encephalitis,

meningoencephalitis and

Stillbirths

In humans, it primarily infects immunocompromised

individuals like

pregnant women,

neonates, and

the elderly

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Listeriosis is a severe foodborne disease characterized by bacteremia and meningoencephalitis in individuals with impaired cell-mediated immunity, Including :

Neonates,

Pregnant woman,

Elderly persons, and

Immunosuppressed patients.

The potential of L. monocytogenes to cause disease correlates with its capacity to survive within macrophages, to invade nonphagocytic cells and replicate therein and also to cross the intestinal, the blood–brain, and the fetoplacental barriers.

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The incidence of listeriosis is rather low, compared to

other common foodborne pathogens such as

Campylobacter species, Salmonella species, Shigella

species, andVibrio species.

However

The outcome is much more severe and often fatal.

In fact, it represents one of the most deadly bacterial

infections due to its high mean mortality rate of 20%–

30%, despite early antibiotic treatment.

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Internalins Proteins (ex : InL A, InL B ,....)

Listeriolysin O (LLO)

Phospholipases

Act A

The Virulence Genes (prfA, PlcA, hly, mpl,...)

PI-PLC

PC-PLC

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There exist two principle mechanisms by which L.

monocytogenes can enter into the host through the

intestinal mucosa.

The first route is direct invasion of the enterocytes

lining the absorptive epithelium of the

microvilli, leading to infection of the intestinal cells

This entry mechanism occurs only in humans and some

susceptible animals.

The second entry pathway is translocation across the

M-cells of Peyer’s patches.

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Internalin (InlA), and InlB were the first surface proteins of L.monocytogenes identified to promote host cell invasion.

They have an amino-terminal leucine-rich repeat domain(LRR) formed by tandem repeats of 20-22 amino acids.

The LRR regions are structurally and functionally important to theninternalization of LM.

LRRs provide versatile recognition units for protein-protein interactions and protein activation in a variety of prokaryotic and eukaryotic proteins.

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L. monocytogenes genome encodes 22 additional proteins

containing LRRs that form the internalin family.

The amino-terminal LRRs are followed by a conserved

inter-repeat domain (IR)

The carboxy-terminal LPXTG motif, which mediates

covalent binding to the cell wall peptidoglycan, is present

in 19 members including the internalin prototype, InlA

One member, InlB, is bound to the lipoteichoic acids on

the bacterial cell wall by electrostatic interactions

involving carboxy-terminal glycine/tryptophan-rich

(GW) modules.

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InlA, an 800-amino-acid protein, is responsible for

bacterial entry into epithelial cells.

InlA specifically interacts with E-cadherin.

E-cadherin: a member of the cadherin superfamily of

calcium-dependent cell adhesion molecules.

E-cadherin is involved in the formation of adherens

junctions in polarized epithelial cells of different tissues.

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The carboxyl terminal of E-cadherin directly interacts

with the intracellular β-catenin.

α-catenin, in turn, binds to β-catenin and interacts with

actin This interaction leads to the formation of a fusion

molecule consisting of the ectodomains of the E-

cadherin and the actin binding site of the α-catenin

which eventually leads to LM entry.

Furthermore, myosin VIIA and its ligand vezatin

together function as the molecular motor in the

internalization of Listeria.

When myosin VIIA binds vezatin, coupled with an actin

polymerization process, it provides the tension

necessary for bacterial internalization.

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InlB, a 630-amino-acid protein, promotes

L. monocytogenes entry into a large variety of mammalian cells including :

Epithelial cells

Endothelial cells

Hepatocytes

Fibroblasts

The hepatocyte growth factor receptor (Met/HGF-R) has

been identified as the major ligand for InlB responsible for

L. monocytogenes entry into non-phagocytic cells, and for

cell scattering and membrane ruffling induced by soluble

InlB

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Met belongs to the family of receptor tyrosine kinases

(RTKs), one of the largest and most important families of

transmembrane signaling receptors expressed by a large

variety of cells.

Met plays crucial roles in :

Organ morphogenesis,

Cell proliferation,

Cell migration and differentiation,

And also in cell growth and invasion during metastasis

in cancer cells

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InlB functionally mimics HGF, the natural Met

ligand, through the binding of its LRRs.

As in the case of the InlA/E-cadherin

interaction, InlB interaction with Met is species

specific: InlB interacts with human and mouse

Met, but does not recognize the guinea-pig or rabbit

receptor.

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Listeriolysin O (LLO), the major and first identified

virulence factor of L. monocytogenes is a member of the

cholesterol-dependent cytolysin (CDC) family of toxins.

These toxins are produced by numerous Gram-positive

bacterial pathogens including :

Streptococcus pneumonia

Bacillus anthracis.

Among these pathogens only L. monocytogenes is known

to infect nonphagocytic cells and LLO, secreted by L.

monocytogenes, is required for bacterial escape from

endocytic vacuoles.

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LLO binds to the host plasma membrane as a monomer

and then forms oligomers composed of up to 50 subunits

that inserts into the plasma membrane forming pores of

about 200e 300 A° diameter.

hly, one of the many genes activated during

infection, leads to the production of Listeriolysin O

(LLO).

Unlike the other CDCs, pore formation by LLO is more

efficient at low pH facilitating the disruption of endocytic

membranes following acidification of the vacuoles.

This is a crucial function for cellular invasion of both

phagocytic and nonphagocytic cells.

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LM also uses phospholipases C to aid in the escape from the vacuole.

Two specific phospholipases (PLCS) are used.

One is the phosphatidylinostiol-specific PLC (PI-PLC), and

The other is more general, phosphatidylinostiol-specific (PC-PLC).

The role of the PI-PLC secreted by LM is to catalyze the production of inositol phosphate and diacylglycerol (DAG) through cleavage of the membrane lipid PI.

DAG then has the ability to activate protein kinase C (PKC).

There are four types of PKCs, but the PKC of the host is shown to be linked with the PI-PLC signaling cascade.

The PKC has been shown to facilitate the permeation of the phagosomal membrane before the bacteria escape.

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In summary, the secretion of PLCs during listerial

infection has several effects on the host cell.

One of these effects has been shown to :

1) Increase the permeation of phagosomal membrane.

2) The activation of PKC through PI-PLC facilitates the

escape of the bacterium.

3) The decreased affinity of L. monocytogenes for the

glycan linker of the GPI-anchored protein due to the

lack or absence of the Vb-strand also increases the

ability for the bacterium to escape during infection.

4) PC-PLC leads to the activation of NF-kB, which allows

the bacteria to exploit the host cell machinery.

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The bacterial surface protein ActA is a major virulence

factor of L. monocytogenes that enables bacterial

propulsion in the cytosol leading to the invasion of yet

uninfected neighboring cells by a process called cell-to-

cell spreading.

Host cell interaction could be mediated by the amino-

terminal region of ActA that has several clusters of

positively charged amino-acids that could bind heparan

sulfate proteoglycans.

The precise mechanism involved in ActA -mediated

invasion remains to be elucidated.

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After infection within the gastrointestinal tract

immediate immune responses are essential for the

control of pathogens, such as L. monocytogenes.

Activation of the innate immune system is triggered

when pathogen-associated molecular patterns (PAMPs)

engage pattern recognition receptors (PRRs) on

intestinal epithelial cells (IECs).

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Typical PAMPs include bacterial carbohydrates, such as

Lipopolysaccharide (LPS)

Mannose

Nucleic acids (both DNA and RNA)

Peptidoglycan components

Lipoteichoic acids, and probably many other

molecules, and are able to trigger the innate immune

response.

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Innate immunity to L. monocytogenes is primarily

mediated by two types of pattern recognition receptors:

The Toll-like receptors (TLRs)

The nucleotide-binding oligomerization domain

(NOD)-like receptors (NLRs).

In addition, there is some experimental evidence for

the involvement of scavenger receptors and a TLR-9

independent cytosolic sensor system for bacterial DNA

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Upon recognition of the presence of microbes

through sensing pathogen-associated molecular

patterns.

TLRs can bind any of the 4 known activating

adaptors:

Myeloid differentiating factor-88 (MyD88)

MyD88 adapter-like (Mal)

TIR domain-containing adapterinducing IFN-ɣ(TRIF)

TRIF-related adapter molecule (TRAM)

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MyD88 appears to be the key adaptor

molecule, because it is required for signaling by all

TLRs with only one exception: TLR3 uses TRIF.

The binding of the activating adaptors results in the

subsequent recruitment of IL-1R, associated kinases

(IRAKs) and downstream activation of transcription

factors including NF-κB and IFN regulatory factor 3

(IRF3), which in turn induces the proinflammatory

cytokines and type I IFNs.

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Toll-like receptor 2 (TLR2) can interact with several

specific ligands, including :

Bacterial lipoproteins

Lipoteichoic

Acids of Gram-positive bacteria such as L.

monocytogenes

Yeast zymosan

TLR2 can form heterodimers with TLR1 and

TLR6, thereby improving the recognition of the target

lipoteichoic acids.

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TLR2 is expressed on the cell surface of intestinal

epithelial cells and its activation by commensal

bacteria is thought to play an important role in the

maintenance of the integrity of the intestinal epithelial

barrier.

TLR2 is also expressed within

phagolysosomes, thus, L. monocytogenes cells that

have escaped into the host cell cytoplasm were not

detected by TLR2.

The importance of TLR2 signaling for early protection

against L. monocytogenes is, however, inconclusive.

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Toll-like receptor 5 (TLR5) can bind to a protein motif

common to the flagellin protein making up the flagella

from many bacteria, such as L. monocytogenes.

TLR5 activation induces NF-κB and stimulates TNF

production, suggesting that TLR5 may serve as a

general alarm system, when the gastrointestinal barrier

is compromised by a broad spectrum of motile bacteria.

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Toll-like receptor 9 (TLR9) recognizes the CpG motifs

present in bacterial DNA. In immune cells, TLR9 is

exclusively localized in the endosomes.

In the intestine, TLR9 was shown to be located on

both, the apical and the basolateral surface of IECs.

Upon activation of TLR9, IκBα is degraded and NF-κB

is activated, again resulting in a proinflammatory

response.

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In conclusion, the available experimental evidence

suggests that TLR2 is the most relevant TLR for

recognition of L. monocytogenes cells However, as

IECs show.

TLR2 commensal ligand-induced activation, TLR2 is

also considered to play an important role in maintaining

the integrity of the intestinal epithelial barrier.

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The NLRs are critical for mucosal innate immunity as

sensors of microbial components and cell injury in the

cytoplasm.

Both NOD1 and NOD2 are important for the innate

immune response against L. monocytogenes, because

they represent intracellular sensors of bacterial

peptidoglycan components that are thought to enter

cells by endocytosis through clathrin-coated pits.

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NOD1 is ubiquitously expressed in adult human

Tissues.

NOD2 is expressed only in leukocytes, DCs, and

epithelial cells.

Activation of NOD1 and NOD2 results in

the translocation of NF-κB and mitogen-activated protein

kinase into the nucleus, to up-regulate the transcription of

proinflammatory genes and mediate antibacterial effects

by the up-regulation of another group of small

antibacterial peptides, the defensins.

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NOD1 recognizes a diaminopimelic acid-containing

dipeptide or tripeptide molecule generated by lysozyme

action on the peptidoglycan of many Gram-negative and

Gram-positive bacteria, including L. monocytogenes.

NOD2 is activated by muramyl dipeptide

(MDP), which is another degradation product of the

peptidoglycan produced by lysozyme and other

(bacterial) peptidoglycan hydrolases.

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DCs respond to different pathogens and initiate the appropriate type of T cell response needed to control the infection.

In response to L. monocytogenes infection, DCs are critical in priming the T cell response, since mice depleted of DCs are unable to generate a CD8 T cell response.

Due to the primarily intracellular localization of L. monocytogenes, CD4 and CD8 T cells mediate most of the adaptive immune response, and are crucial for long-termimmunity after initial L. monocytogenes infection.

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Almost any cell type that harbors L. monocytogenes in the cytoplasm can process the proteins secreted from the pathogen, by degradation and subsequent loading on MHC class I molecules, in order to present them on the cell surface to CD8 T cells.

Only professional antigen presenting cells (APCs) can present antigens derived from lysosomal degradation via the MHC class II pathway to CD4 T cells.

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The CD8 T cells mediate the anti-Listeria immunity by

two synergistic mechanisms:

first:

by secretion of IFN-γ to activate macrophages;

Secondly:

by lysis of infected cells via perforin and

granzymes, leading to the exposure of intracellular

bacteria to the activated macrophages

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IFN-γ is known to be essential for host resistance to

intracellular pathogens such as L. monocytogenes, as it

mediates the activation of resting macrophages that

more efficiently restricts the multiplication of

intracellular pathogens and promotes long-term

protective cellular immunity.

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L. monocytogenes induces a strong T-helper type 1

response and, similar to CD8 T cells, CD4 T cells

also secrete IFN-γ.

The strong CD8 and CD4 T cell responses results in a

stable population of memory T cells specific for L.

monocytogenes

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In the intestine, NKT cells (lymphocytes expressing

both NK and T cell markers) play an important role in

the control of early infection with L. monocytogenes.

In general, adaptive immune responses in the intestine

are characterized by high numbers of IgA producing

plasma cells, regulatory T cells, and IL-17 producing T

cells whose development is closely linked to factors

produced by PRRs expressing IECs, DCs, and

macrophages.

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