molecular details of serum resistance of yersinia ... · molecular details of serum resistance of...

Molecular Details of Serum Resistance

of Yersinia enterocolitica

Marta Biedzka-Sarek

Department of Bacteriology and ImmunologyHaartman Institute

Faculty of Medicine

and

Helsinki Graduate School of Biotechnology and Molecular Biology

University of HelsinkiFinland

ACADEMIC DISSERTATION

To be publicly discussed with the permission of the Faculty of Medicine of theUniversity of Helsinki, for public criticism in the Seth Wichmann Hall of the

Naistenklinikka, Haartmaninkatu 2, on 5th December 2008, at 12 noon

Supervisor

Mikael Skurnik

Professor of Bacteriology, PhDDepartment of Bacteriology and ImmunologyHaartman InstituteUniversity of HelsinkiFinland

Reviewers

Benita Westerlund-Wikström

Docent, PhDDepartment of Biological and Environmental SciencesGeneral MicrobiologyUniversity of HelsinkiFinland

and

Ilkka Julkunen

Professor, MD, PhDDepartment of Viral Diseases and ImmunologyNational Public Health InstituteHelsinki, Finland

Oponent

Jürgen Heesemann

Professor, MD, PhDMax von Pettenkofer Institut of Hygiene and Medicinal MicrobiologyMunich, Germany

Cover figure “Pyramide” by Ayman Abdo

ISBN 978-952-92-4685-4 (paperback)ISBN 978-952-10-5092-3 (pdf)Printed at Yliopistopaino, Helsinki, Finland, 2008

To my family

CONTENTS

LIST OF ORIGINAL PUBLICATIONS .....................................................................................................1

ABBREVIATIONS .........................................................................................................................................1

ABSTRACT .....................................................................................................................................................1

1. REVIEW OF THE LITERATURE..........................................................................................................3

1.1 INTRODUCTION .......................................................................................................................................31.2 SERUM BACTERICIDAL ACTIVITY – DISCOVERY AND OVERVIEW OF THE COMPLEMENT SYSTEM ........4

1.2.1 Classical pathway (CP)..................................................................................................................61.2.2 Alternative pathway (AP) ..............................................................................................................81.2.3 Lectin pathway (LP) ......................................................................................................................81.2.4. Lytic pathway................................................................................................................................9

1.3 COMPLEMENT REGULATION .................................................................................................................111.3.1 Fluid-phase complement regulators ............................................................................................11

1.3.1.1 Factor I................................................................................................................................................... 111.3.1.2 Factor H protein family......................................................................................................................... 13

1.3.1.2.1 Factor H ........................................................................................................................................ 131.3.1.2.2 Factor H-like protein (FHL-1) ..................................................................................................... 151.3.1.2.3 Factor H-related proteins (FHR).................................................................................................. 15

1.3.1.3 C4b-binding protein .............................................................................................................................. 161.3.2 Membrane-bound complement regulators ..................................................................................17

1.4 FUNCTIONS OF THE COMPLEMENT SYSTEM..........................................................................................171.4.1 Opsonin-mediated clearance of microbes and host debris .........................................................171.4.2 Direct lysis of pathogens..............................................................................................................191.4.3 Induction of inflammation ...........................................................................................................191.4.4 Shaping the specific immune response .......................................................................................19

1.5 BACTERIAL MECHANISMS OF COMPLEMENT EVASION.........................................................................201.5.1 Interference with initial complement activation .........................................................................20

1.5.1.1 Binding of immunoglobulins via Fc portion........................................................................................ 201.5.1.2 Immunoglobulin and C3/C3b degradation........................................................................................... 211.5.1.3 Binding of C1 inhibitor or C1q inhibition ........................................................................................... 21

1.5.2 Inhibition of C3 convertases........................................................................................................211.5.2.1 Microbial factors directly interfering with C3 convertases................................................................. 211.5.2.2 Microbial factors mimicking FI-cofactors to accelerate decay of the C3 convertases ...................... 221.5.2.3 Microbial factors binding host complement regulators....................................................................... 22

1.5.3 Interference with MAC formation (terminal pathway) ..............................................................241.5.3.1 Physical barriers .................................................................................................................................... 241.5.3.2 Microbial proteins interfering with membrane attack complex .......................................................... 251.5.3.3 Acquisition of host terminal pathway regulators ................................................................................. 25

1.5.4 Interference with generation and function of complement-derived chemoattractants ..............251.6 GENUS YERSINIA....................................................................................................................................26

1.6.1 Y. pestis.........................................................................................................................................261.6.2 Enteropathogenic Yersiniae .........................................................................................................27

1.6.2.1 Y. pseudotuberculosis ........................................................................................................................... 281.6.2.2 Y. enterocolitica .................................................................................................................................... 29

1.6.3 Virulence determinants of pathogenic Yersiniae........................................................................301.6.4 Y. enterocolitica surface factors shaping serum resistance phenotype ......................................36

1.6.4.1 YadA...................................................................................................................................................... 361.6.4.1.2 YadA-mediated serum resistance ................................................................................................ 38

1.6.4.2 Ail .......................................................................................................................................................... 401.6.4.2.1 Ail-mediated serum resistance ..................................................................................................... 40

1.6.4.3 LPS ........................................................................................................................................................ 411.6.4.3.1 O-antigen ...................................................................................................................................... 411.6.4.3.2 Outer core ..................................................................................................................................... 431.6.4.3.3 LPS and serum resistance............................................................................................................. 44

2. AIMS OF THE STUDY............................................................................................................................45

3. MATERIALS AND METHODS.............................................................................................................46

4. RESULTS AND DISCUSSION...............................................................................................................55

4.1 COMBINED ACTION OF Y. ENTEROCOLITICA OUTER MEMBRANE PROTEINS AND LPS IN RESISTANCE

TO COMPLEMENT-MEDIATED LYSIS......................................................................................................554.2 Y. ENTEROCOLITICA AND ACQUISITION OF COMPLEMENT REGULATORS..............................................57

4.2.1 Acquisition of FH.........................................................................................................................574.2.2 Acquisition of C4bp.....................................................................................................................59

4.3 IDENTIFICATION OF FH AND C4BP RECEPTORS ON Y. ENTEROCOLITICA .............................................604.3.1 Characterization of YadA- Ail- mediated interactions with FH and C4bp ...............................62

4.3.1.1 YadA and FH ........................................................................................................................................ 624.3.1.1.1 Mapping of YadA regions involved in FH-binding.................................................................... 63

4.3.1.2 YadA and C4bp..................................................................................................................................... 664.3.1.3 Ail and FH ............................................................................................................................................. 67

4.3.1.3.1 Mapping Ail regions involved in FH binding ............................................................................. 684.3.1.4 Ail and C4bp ......................................................................................................................................... 69

5. CONCLUSIONS AND FUTURE PROSPECTS...................................................................................70

ACKNOWLEDGEMENTS .........................................................................................................................72

BIBLIOGRAPHY .........................................................................................................................................75

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on following original publications, which are referred to in the text

by their Roman numerals.

I. Biedzka-Sarek M, Venho R, Skurnik M. Role of YadA, Ail, and

lipopolysaccharide in serum resistance of Yersinia enterocolitica serotype

O:3. Infection and Immunity 2005 Apr; 73(4):2232-44.

II. Skurnik M, Biedzka-Sarek M, Lubeck PS, Blom T, Bengoechea JA,

Perez-Gutierrez C, Ahrens P, Hoorfar J. Characterization and biological

role of the O-polysaccharide gene cluster of Yersinia enterocolitica

serotype O:9. Journal of Bacteriology 2007 Oct; 189(20):7244-53.

III. Biedzka-Sarek M, Jarva H, Hyytiäinen H, Meri S, and Skurnik M.

Characterization of complement factor H binding to Yersinia enterocolitica

serotype O:3. Infection and Immunity 2008 Sep; 76(9):4100-9.

IV. Biedzka-Sarek M, Salmenlinna S, Gruber M, Lupas A, and Skurnik M.

Functional mapping of YadA- and Ail-mediated binding of human factor H

to Yersinia enterocolitica serotype O:3. Infection and Immunity 2008 Nov;

76(11):5016-27

V. Kirjavainen V�, Jarva H�, Biedzka-Sarek M�, Blom A, Skurnik M, and

Meri S. Yersinia enterocolitica serum resistance proteins YadA and Ail

bind the complement regulator C4b-binding protein. PLoS Pathogens 2008

Aug; 4(8), � these authors contributed equally

These articles have been reprinted with the permissions of the copywrite holders.

ABBREVIATIONS

ABBREVIATIONS

aa amino acid(s)

aHUS atypical hemolytic uremic syndrom

Ail attachment and invasion locus

AP alternative pathway

bp base pair(s)

BSA bovine serum albumin

C1-INH C1 inhibitor

C4bp C4b-binding protein

CCP complement control protein

Clm chloramphenicol

CP classical pathway

CR1 complement receptor type 1

DAF decay-accelerating factor

DNA deoxyribonucleic acid

EGTA ethylene glycol-bis (�-aminoethyl ether)-N,N,N',N'- tetraacetic acid

ELISA enzyme-linked immunoadsorbent assay

FH factor H

FHL-1 FH-like protein 1

FHR FH-related protein

FI factor I

hms hemin storage locus

HPI high-pathogenicity island

IC inner core of LPS

kb kilo base (1000 base pairs)

kDa kilodalton = 1.660 � 10-21 g

Km kanamycin

LP lectin pathway

LPS lipopolysaccharide

mAb monoclonal antibody

MAC membrane attack complex

MASP mannose-binding lectin-associated serine protease

MBL mannose-binding lectin

MCP membrane cofactor protein

MPGN membranoproliferative glomerulonephritis

NDP nucleotide diphosphate

NHS normal human serum

O-ag O-antigen

OC outer core of LPS

PAGE polyacrylamide gel electrophoresis

ABBREVIATIONS

PCR polymerase chain reaction

pYV Yersinia virulence plasmid

RCA regulators of complement activation

SCR short consensus repeat

SDS sodium dodecyl sulphate

Tet tetracyclin

Tx-114 Triton X-114

Und-P undecaprenyl phosphate

YadA Yersinia adhesin A

Yops Yersinia outer proteins

Yst Yersinia heat-stable toxin

ABSTRACT

- 1 -

ABSTRACT

The complement system, consisting of a set of plasma and membrane-bound proteins,

serves as an essential part of the human immune defense. Its activation (through the

classical, lectin, and alternative pathways) tags microbes for destruction by phagocytic

cells or even leads to microbial lysis. In addition, anaphylatoxins generated during

complement activation can trigger a range of pro-inflammatory responses. This

antimicrobial action of the complement system can, however, be evaded by many

microbes. Pathogens have developed a number of complement-evasion strategies, one

of which is the recruitment of serum proteins called complement regulators. These

regulators protect host tissues against excessive complement-mediated damage and are

present in plasma in high concentrations. This, however, makes them an easy target for

pathogens. Acquisition of proteins such as factor H (FH) and C4b-binding protein

(C4bp) provides microbes with the means to inhibit complement activation on their

surfaces.

Yersinia enterocolitica is a Gram-negative enteropathogen that resists bactericidal

action in human serum. This bacterium causes gastroenteritis and as post-infection

sequelae reactive arthritis, myocarditis, erythema nodosum, and glomerulonephritis.

The aim of the present study was to characterize complement evasion strategies of Y.

enterocolitica and to identify bacterial surface factors associated with this

phenomenon.

This thesis reveals that the Y. enterocolitica serotype O:3 adhesins, YadA and Ail, are

the most crucial serum resistance factors (I). Although the lipopolysaccharide (LPS) of

this serotype was not found to participate in serum resistance, serotype O:9 LPS

strengthened YadA-mediated resistance (I, II). Differences in serum resistance

between different Y. enterocolitica serotypes can thus be expected.

Y. enterocolitica was found to bind FH, a fluid-phase inhibitor of the alternative

complement pathway (III). The receptors for FH on the Y. enterocolitica surface were

mapped to YadA and Ail (III). Ail was found to acquire FH only in the absence of the

LPS O-antigen (O-ag) and the outer core (OC), whereas YadA binds the regulators

also in the presence of LPS (III). In addition, YadA appears to bind to short consensus

repeats (SCRs) of the entire polypeptide chain of FH, whereas Ail targets SCRs 6 and

ABSTRACT

- 2 -

7 (III). Importantly, both YadA- and Ail-bound FH is fully functional and displays

cofactor activity for factor I (FI)-mediated cleavage of C3b (III). YadA is a lollipop-

shaped outer membrane protein that consists of a head, a neck, and a coiled-coil stalk

domain. FH targets multiple higher structural and discontinuous motifs of the YadA

stalk (IV).

Another complement regulator shown to be acquired by Y. enterocolitica is C4bp (V).

C4bp retains its function when bound to the bacterial surface, and YadA and Ail

function as C4bp receptors (V). Similar to FH binding, however, Ail acquires C4bp

only in the absence of the LPS O-ag and OC (V). C4bp binding to Ail was likely

hydrophobic, but that to YadA, ionic in nature (V).

Taken together, these data provide evidence that Y. enterocolitica recruits the

complement regulators FH and C4bp. Thus, Y. enterocolitica belongs to a constantly

growing group of pathogens utilizing the most widely disseminated mechanism of

complement evasion.

REVIEW OF THE LITERATURE

- 3 -

1. REVIEW OF THE LITERATURE

1.1 Introduction

Human beings provide a home for a myriad of microbes. Many microbes produce

vitamins and nutrients, ferment food, break down toxic chemicals, and protect us from

pathogenic microbes which we encounter every day. The latter, however, often find

ways to cause infection, exploiting a wide range of strategies to penetrate physical

barriers such as skin or mucous membranes and to survive and persist in the host. The

first obstacle they must combat is the action of the non-specific innate immune system.

One of its essential arms is the complement system, the first line of defense activated

immediately upon pathogen entry. The importance of the complement system in host

defense against invading pathogens is reflected by increased susceptibility to microbial

infection of individuals deficient in certain complement components. Complement is

bactericidal against Gram-negative bacteria, it acts as an opsonin, and its cleavage

products contribute to induction of inflammation.

That pathogens adapt quickly to environmental changes makes them tenacious

opponents. They express surface factors to manipulate the host complement system

and avoid complement-mediated recognition and eradication. Studying microbial

factors conferring complement-resistance at molecular level may provide means for

their neutralization, and this could contribute to the prevention and treatment of

microbial infections.


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1.2 Serum bactericidal activity – discovery and overview of the complement

system

The complement story, as reviewed by Lachmann (Lachmann, 2006), began in the late

1880’s when von Fodor, Nuttall, and Buchner independently observed the bactericidal

activity of normal serum. Discovery of the complement was, however, carried out by

Bordet (1870-1961), who received the Nobel Prize in Medicine in 1919. In 1895

Bordet showed the lysis of Vibrio cholerae by a mixture of normal and heated immune

sera. Thereby he demonstrated the importance of both for bacterial lysis: the heat-

labile “activity” of serum (termed by Ehrlich complement), and the heat-stable

antibody. Six years later, together with Gengou, Bordet published the first and seminal

report on complement fixation, in which he described the ability of antigen-antibody

complexes to remove complement activity from serum. Complement was regarded as a

single substance until 1907 when Ehrlich and Brand fractionated serum and showed

that the heat-labile complement proteins C1 and C2 act sequentially and are required

for complement activity. The first heat-stable complement component identified was

C3, successfully removed from serum by use of cobra venom by Ritz in 1912, and

yeast cells by Coca in 1914. Although C4 was described by Gordon in 1926, the

correct sequence in which these four complement components reacted

(C1�C4�C2�C3) was only established 30 years later by Ecker’s group. Until the

late 1950’s complement function was connected exclusively to complementing the

ability of the antibodies to cause a target lysis. Then Louis Pillemer suggested a

properdin-dependent but antibody-independent pathway of C activation. This

“alternative pathway” and its distinct protein constituents were fully elucidated and

accepted by the scientific community only in the 1970`s. At that time, the lytic activity

of C was shown to involve at least 11 different proteins. In recent years, a “lectin

pathway” of C activation, functioning through recognition of mannan polysaccharides

on microbial pathogens has been identified [the entire history is reviewed in

(Lachmann, 2006)].

These initial studies regarding complement were, therefore, dealing with bactericidal

activity against antibody-coated Gram-negative bacteria. Today, in addition to this

function, complement is known to act as an opsonin and an inducer of inflammation.

Its opsonin activity results in enhanced phagocytosis of the complement-tagged

microbes whereas its inflammatory activity is related to the attraction of immune cells


- 5 -

along the gradient of complement cleavage products. The complement system

comprises approximately 35 fluid-phase (Table 1) and membrane-bound proteins. It is

activated via the classical, the alternative, and the lectin pathways, which sense

microbes by means of different recognition molecules. Once the complement is

activated, a cascade of proteolytic reactions leads to the cleavage of C3, which is a

converging stage for all three pathways. Subsequently, C3 cleavage is followed by the

lytic pathway and the generation of the membrane attack complex (MAC). MAC

forms a pore in the target cell and in consequence disrupts the membrane, causing the

cell death.

Table 1. Components of the complement system.

Component Structure Plasma conc.

(μg/ml)

Classical pathway

C1 3 subunits: C1q (460kDa), C1r (80kDa),C1s (80kDa) in a complex (C1qr2s2)

180

C4 3 chains (�, 97 kDa; �, 75 kDa; �, 33 kDa) 600

C2 single chain, 102 kDa 20

Alternative pathway

Factor B single chain, 93 kDa 210

Factor D single chain, 24 kDa 2

Properdin oligomers of 53-kDa chains 5

Lectin pathway

MBL polymer of 32-kDa chains 0-5

MASP-1 single chain, 100 kDa 1.5-12

MASP-2 single chain, 76 kDa ND

Common:

C3 2 chains: �, 110 kDa; �, 75 kDa 1300

Terminal pathway

C5 2 chains: 115 kDa, 75 kDa 70



C8 3 chains: �, 65 kDa; �, 65 kDa; �, 22 kDa 55


MBL, mannose-binding lectin; MASP, mannose-binding lectin-associated serine protease; ND, non-detectable


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1.2.1 Classical pathway (CP)

Binding of antibody to its specific antigen on the surface of a microbe opens up the

binding sites in the bound antibody for the classical pathway pattern recognition

molecule C1q. C1q, a 460-kDa multimeric protein, resembles in structure a bouquet of

six tulips with globular “head” domains and collagen-like triple-helical “stems”

(Calcott and Müller-Eberhard, 1972; Reid and Porter, 1976; Schumaker et al., 1982;

Strang et al., 1982). In the presence of Ca2+, C1q, together with two C1r and two C1s

serine protease molecules (C1qr2s2), forms the C1 complex (Busby and Ingham, 1990;

Loos et al., 1980; Thielens et al., 1990). Six globular heads of the C1q subunits

recognize Fc regions of IgM or multiple IgG molecules only when they are deposited

on a microbial surface (Ishizaka et al., 1966; Schumaker et al., 1986). C1q can also

activate the classical pathway via binding to C-reactive protein (CRP), nucleic acids,

LPS and immune complexes (Jiang et al., 1992a, 1992b; Loos, 1982; Nauta et al.,

2002).

C1q binding to a target causes a mechanical stress transmitted from the C1q stems to

C1r molecules (Gaboriaud et al., 2004), which in turn activates two molecules of C1s.

C1s first targets the C4 molecule (Patrick et al., 1970). C4 comprises three chains

linked together by disulfide bridges and an internal thioester group which upon

disruption mediates a covalent attachment to a microbial surface (Fig. 2) (Dodds et al.,

1996; Janatova and Tack, 1981; Schreiber and Müller-Eberhard, 1974). C1s-mediated

cleavage of C4 generates two fragments: a small C4a acting as an anaphylatoxin

(Gorski et al., 1979) and a larger C4b. C4b binds to the microbial surface, since the

exposed thioester group is prone to hydrolysis (Law and Dodds, 1990, 1997), and acts

as the C2 receptor in the presence of Mg2+ (Fig. 1). C2 becomes susceptible to C1s and

is cleaved into C2a and C2b (Nagasawa and Stroud, 1977). C2a remains complexed

with C4b, whereas C2b is released in the microenvironment. C4b2a complex is known

as the classical pathway C3 convertase (Kerr, 1980), in which the catalytic subunit C2a

cleaves C3 (Fig. 1).


- 7 -

C3 is the most abundant complement protein in serum. It consists of a 119-kDa �-

chain and a 75-kDa �-chain linked together by a single disulfide bond and non-

covalent forces (Fig. 2) (Lambris, 1988). Similarly to C4, C3 possesses an internal

thioester bond hidden within a hydrophobic pocket in native C3 (Janssen et al., 2006;

Law and Dodds, 1990, 1997). Upon C3 cleavage at a single site on the �-chain, C3a,

called anaphylatoxin, is released into the environment (Hugli, 1975), while C3b binds

to nearby surfaces via an exposed thioester (Law and Levine, 1977; Law and Dodds,

1997; Sim et al., 1981). Some of the C3b binds to CP C3 convertase (C4b2a) to form

C4b2a3b complex, which is termed the CP C5 convertase (Fig. 1) (Takata et al.,

1987).

Alternative PathwayClassical Pathway

C1

Lectin Pathway

MBL-MASP C3b

C4

C4bC2

C4b2a

(C3 convertase)

C4

C4bC2

C3bB

FactorD

Properdin

C3bBb

(C3 convertase)

C3 C3C3b

C4b2a3b

(C5 convertase)

C3bBb3b

(C5 convertase)

C5 C5C5b

MAC

C6-C9

Figure 1. Schematic overview of the complement activation cascade (modified from Miwa andSong, 2001).


- 8 -

1.2.2 Alternative pathway (AP)

The activation of the alternative complement pathway begins with the activation of C3

and it requires factors B and D, properdin, and Mg2+, all of which are present in

normal serum (Fig. 1). The classical pathway is not the only source of the C3b

participating in activation of the alternative pathway. C3b is also generated

spontaneously by hydrolysis of the internal thioester bond in the native fluid phase C3

(tick-over mechanism) (Lachmann and Hughes-Jones, 1984). Native C3 is hydrolyzed

to an active intermediate known as C3(H2O) (Pangburn et al., 1981). C3(H2O) binds

factor B, making the latter susceptible to cleavage by factor D (Lesavre and Müller-

Eberhard, 1978). Factor D is a serine protease and cleaves Factor B into Ba and Bb.

Generated C3(H2O)Bb complex acts as the fluid phase alternative pathway C3

convertase. Fluid phase C3b, generated by the convertase, is deposited on a bacterial

surface via its thioester group exposed on that surface (Law and Dodds, 1990, 1997).

In the presence of Mg2+ it forms a complex with factor B (Fig. 1). The resulting C3bB

is activated to become AP C3 convertase, C3bBb, by factor D and is stabilized by

properdin (Fig. 1).

AP convertases are crucial for the C3b amplification loop (Fig. 3). The serine protease

Bb of the AP convertase very efficiently degrades C3, generating large amounts of

C3b, which in turn can become deposited on nearby surfaces or contribute to C5

convertase formation via binding to C3 convertases of AP and CP. In consequence, the

AP amplifies activation of the CP (Lachmann and Hughes-Jones, 1984).

1.2.3 Lectin pathway (LP)

The C4 activation can also be achieved without the participation of the antibodies and

C1 via the lectin pathway. This pathway is initiated by MBL and the MBL-associated

serine proteases: MASP-1 and MASP-2 (Sato et al., 1994; Thiel et al., 1997). MBL

binds to certain mannose residues on many bacteria and subsequently interacts with

MASP-1 and MASP-2. The MBL-MASP1-MASP2 complex is analogous to the C1

complex of the classical pathway. A major difference is, however, that C1 binds solely

to a target tagged with specific antibodies, while MBL binds directly to mannose- or

N-acetyl-glucosamine-rich polysaccharides commonly presented by a wide range of

microorganisms (Jack et al., 2001). Upon activation, the MASPs cleave the

downstream complement components C4 and C2 (Matsushita and Fujita, 1992; Thiel


- 9 -

et al., 1997). From this point onwards, the lectin pathway is identical to the classical

pathway (Fig. 1).

1.2.4. Lytic pathway

The classical, lectin, and alternative pathways end with the formation of C5 convertase

that then leads to the assembly of MAC via the lytic pathway. C5 convertases

(C3b4b2a, C3bBbC3b) generated by one of the pathways cleave C5 (Fig. 1) (Cooper

and Müller-Eberhard, 1970; DiScipio et al., 1983). C5 comprises two chains linked

together with one disulfide bond. C5, however, lacks the internal thioester group

characteristic of C3 and C4 (DiScipio et al., 1983). C5 cleavage generates two

biologically active fragments: C5a, a potent chemokine, and anaphylatoxin (Cochrane

and Müller-Eberhard, 1968; Ward and Newman, 1969), and C5b, an initial constituent

in MAC assembly (Fig. 1).

C5b is very labile and needs to bind to C6 very rapidly (DiScipio et al., 1983). C5b6

subsequently binds C7 to yield the hydrophobic C5b67 complex (DiScipio et al., 1988;

DiScipio, 1992). The C5b67 complex attaches to nearby membranes without harming

the cells. Upon C8 binding, the resulting C5b678 complex inserts deeply into the

membrane (Kolb and Müller-Eberhard, 1973; Tamura et al., 1972) and subsequently

binds C9, causing its nonenzymatic polymerization (Tschopp et al., 1982b). In

consequence, C9 undergoes hydrophilic-amphiphilic and conformational transition to a

tubular structure (Tschopp et al., 1982a). Acquisition of C9 molecules leads to the

formation of a pore-like structure of MAC which passes through the membrane

(Tschopp et al., 1982a). The pore is approximately110 Å wide (Podack and Tschopp,

1982) and has a hydrophilic lumen through which, according to the “doughnut theory”,

cell contents leak out and water is drawn inside, generating the osmotic pressure which

causes rupture of cellular membranes (Lachmann, 2006). In addition, leakage through

the pore may be assisted by passage of the solutes and water through the “leaky

patches” formed in the membrane upon MAC insertion (Lachmann, 2006).


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Table 2. Fluid-phase regulators of the complement system

Regulator Size

(kDa)

Serum

conc.

(μg/ml)

Function Reference

C1 inhibitor

(C1-INH)

105 200 blocks active sites on C1s ans C1r orremoves them from the C1 complex

prevents C1 autoactivation

inhibits formation of AP C3

convertase

Harrison, 1983;

Harpel and Cooper,

1975; Ziccardi and

Cooper, 1979;

Ziccardi, 1982, 1985;

Jiang et al., 2001

FH 150 500 accelerates decay of AP C3convertase

inhibits formation of AP C3convertase

cofactor for FI

Nilsson and Müller-

Eberhard, 1965;

Pangburn et al.,

1977; Weiler et al.,

1976; Whaley and

Ruddy, 1976

C4bp 570 200 accelerates decay of CP/LP C3

convertase

inhibits formation of CP/LP C3

convertase

cofactor for FI

Barnum, 1991; Gigli

et al., 1979;

Dahlbäck, 1983

FI 90 30 cleaves C3b and C4b in the

presence of cofactor

Ruddy and Austen,

1969; Ahearn and

Fearon, 1989

Properdin 210 26 stabilizes AP C3 convertase Fearon and Austen,

1975; Medicus et al.,

1976; Smith et al.,

1984

Clusterin

(SP40,40)(Apolipoprotein

J)

70 50 prevents MAC insertion by binding to

terminal complement complexesC5b-7, C5b-8, and C5b-9

Tsuruta et al., 1990;

Choi et al., 1989;

Murphy et al., 1989;

Tschopp et al., 1993

Vitronectin

(S-protein)

75 500 prevents MAC insertion by binding toterminal complement complexes

C5b-7

Podack and Müller-

Eberhard, 1979


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1.3 Complement regulation

1.3.1 Fluid-phase complement regulators

Tight regulation of the complement system crucial in preventing bystander injury of

host cells and systemic depletion of complement, is provided by fluid phase

complement regulators (Table 2). These regulate complement activation at C1 (C1

inhibitor), C3 (factor H, C4b-binding protein, properdin) or MAC (clusterin,

vitronectin) levels. The complement inhibitors that act at the C3 level function as

cofactors for serine protease FI-mediated cleavage of C3b and C4b. In this way, they

prevent formation of C3 convertases or downregulate their activity. The latter

function, termed decay-accelerating activity, relies on displacing the C3 convertase

enzymatic subunits C2a or Bb.

1.3.1.1 Factor I

FI is a 90-kDa (heavy chain, 50 kDa; light chain, 38 kDa) plasma protease which

circulates in its active form at a concentration of �30 μg/ml (Ruddy and Austen, 1969).

It cleaves arginyl bonds of its substrates C3b and C4b only when these are complexed

to one of the complement regulators. Membrane cofactor protein (MCP), complement

receptor type 1 (CR1), and C4bp contribute to the cleavage of both C3b and C4b,

whereas FH exclusively participates in the cleavage of C3b (Ahearn and Fearon, 1989;

Blom et al., 2003; Fujita and Nussenzweig, 1979; Pangburn et al., 1977; Seya et al.,

1986).

Inactivation of C3b by FI can occur at three sites of the �’-chain and results in the

generation of iC3b1, iC3b2, C3c or C3dg (Fig. 2A) (Davis and Harrison, 1982; Lambris

et al., 1996; Medicus et al., 1983; Ross et al., 1982). The latter two products are

generated when CR1 serves as a co-factor (Ahearn and Fearon, 1989). FI-mediated

inactivation of C4b targets two sites of the �-chain and results in the generation of C4c

and C4d fragments (Fig. 2B).


- 12 -

119 kDa

S–C=O

S–S

75 kDa

S S

�

�

C3

C3b9

C3a

iC3b2

27 kDa 41 kDa

C3dg

C3c

C3 convertase

FI + cofactor

(thioester)

110 kDa

R-O–C=O

S–S

75 kDa

S S

R-O–C=O

S–S

75 kDa

S S

67 kDa 41 kDa

R-O–C=O

FI + cofactorR-O–C=O

S–S

75 kDa

S S

67 kDa 43 kDa

iC3b1

S–S

75 kDa

S S

40 kDaFI+CR1

C3f

93 kDa�

�

C4

S–S

32 kDa

S S

�

85 kDa

S–S

75 kDa

S–S

32 kDa

S S

C1s

8

C4a

S–C=OS–S

75 kDa

(thioester)

R-O–C=O

25 kDa

S–S

75 kDa

S–S

32 kDa

S S

R-O–C=O

45 kDa15 kDa

C4b

FI+ cofactor

C4d

C4c

Figure 2. Schematic representation of C3 (A) and C4 (B) cleavage sites (modified fromMorgan, 2000; Sahu and Lambris, 2001)

A.

B.


- 13 -

1.3.1.2 Factor H protein family

1.3.1.2.1 Factor H

FH was identified as a �1H globulin by Nilsson and Müeller-Eberhard (Nilsson and

Müller-Eberhard, 1965). It is a soluble single-chain glycoprotein of 150 kDa present in

plasma at a concentration of �500 μg/ml. FH accelerates the decay of the AP C3

convertase, competes with FB for C3b binding, and acts as a cofactor for FI-mediated

C3b inactivation (Fig. 3) (Pangburn et al., 1977; Weiler et al., 1976; Whaley and

Ruddy, 1976). In this way, FH downregulates complement in the fluid phase and on

cell surfaces.

FH distinction of self from non-self (AP activators) surfaces is based on recognition of

polyanionic substances such as sialic acids and glycosaminoglycans (Fearon, 1978;

Meri and Pangburn, 1990). These coat the host cells but, in general, do not cover

foreign cell membranes. High-affinity interaction between FH and C3b occurs when

the latter is accompanied by polyanions and results in the inhibition of AP. In the

Figure 3. FH and C4bp control steps. Solid lines, complement activation; dashed lines,complement inhibition


- 14 -

absence of polyanions, however, FH affinity for C3b dramatically decreases, and in

consequence AP activation proceeds (Kazatchkine et al., 1979; Pangburn et al., 1983).

FH is composed of 20 SCRs joined by short linker regions of 3-8 amino acids. SCRs

are arranged in a continuous fashion and resemble a string of beads (Discipio, 1992;

Ripoche et al., 1988). SCRs 1-4, SCRs 12-14, and SCRs 19-20 provide the binding

sites for C3b, whereas SCR7, SCR 13, and SCRs 19-20 bind heparin and polyanions

(Alsenz et al., 1985; Blackmore et al., 1996, 1998b; Gordon et al., 1995; Jokiranta et

al., 1996, 2000; Kühn et al., 1995; Pangburn et al., 1991; Prodinger et al., 1998;

Sharma and Pangburn, 1996). Decay-accelerating and cofactor functions have been

ascribed to SCRs 1-4 (Gordon et al., 1995; Kühn et al., 1995; Kühn and Zipfel, 1996)

(Fig. 4).

Deficiency of FH in humans has been associated with membranoproliferative

glomerulonephritis (MPGN) and atypical hemolytic uremic syndrom (aHUS).

Complete FH deficiency is, however, a rare phenomenon (Reis et al., 2006). Missense

mutations clustered within SCRs 19-20 have been exclusively linked to aHUS (Ault,

2000; Dragon-Durey et al., 2004; Jokiranta et al., 2006; Manuelian et al., 2003;

Figure 4. FH protein family (modified from Jozsi et al., 2005; Zipfel et al., 2002). SCRs arealigned according to regions of homology. Binding sites for C3b and heparin are shown forFH and FHL-1.


- 15 -

Neumann et al., 2003; Zipfel, 2001), a rapidly progressing renal disease that manifests

as an endothelial cell damage, hemolysis, massive AP activation, and kidney failure

(Ruggenenti et al., 2001). As the C terminus is crucial for binding to C3b, C3d,

heparin, and polyanions, mutation within the C-terminal SCRs abolishes critical FH

functions for complement regulation.

1.3.1.2.2 Factor H-like protein (FHL-1)

FHL-1 (reconectin) is an alternatively spliced product of the FH-encoding gene

(Estaller et al., 1991). It comprises seven SCRs, being identical to the first seven N-

terminal SCRs of FH and the unique C-terminal sequence of four amino acids (Fig. 4).

Thus, FHL-1, similar to FH, displays cofactor and decay-accelerating activities and

possibly contributes to complement regulation in vivo (Kühn and Zipfel, 1996; Zipfel

and Skerka, 1999). However, the plasma concentration of this 42 kDa protein is

approximately one-tenth that of FH.

1.3.1.2.3 Factor H-related proteins (FHR)

FHRs are encoded by separate genes located within the regulators of complement

activation (RCA) gene cluster on chromosome 1 (Zipfel et al., 1999). To date, five

human FHRs have been identified, and these share structural similarities with FH (Fig.

4). FHR-1 consists of five SCRs and is found in the 37-kDa (FHR1-�) or 43-kDa

(FHR1-�) forms with one or two carbohydrate-side residues, respectively (Estaller et

al., 1991; Skerka et al., 1991). FHR-2 is composed of four SCRs and exists in a

glycosylated (29-kDa) or in a non-glycosylated (24-kDa) form (Skerka et al., 1992).

FHR-3 and FHR-4B are similar to each other and are organized into five SCRs. FHR-3

occurs in several glycosylated forms in human plasma (ranging from 35 kDa to 56

kDa), whereas FHR-4B, a 42-kDa protein, is one of the two FHR-4 gene products

derived by means of alternative splicing (Jozsi et al., 2005; Skerka et al., 1993, 1997).

The other product of the FHR-4 gene is FHR-4A. This glycosylated protein of 86 kDa

consists of nine SCRs, due to duplication of four SCRs (Fig. 4) (Jozsi et al., 2005).

FHR 5 contains nine SCRs homologous to those of FH (McRae et al., 2001).

The structural similarity between FHR proteins and FH suggests that FHRs share some

of the FH activities. Indeed, FHR-3, FHR-4, and FHR-5 have been shown to bind C3b

while FHR-1, FHR-3, and FHR-5 bind heparin (Hellwage et al., 1999; McRae et al.,


- 16 -

2001, 2005; Prodinger et al., 1998). In addition, FHR-3 and FHR-4 have been

suggested to modify the conformation of C3b to enhance FH cofactor activity

(Hellwage et al., 1999). FHR-5 is a relatively weak cofactor for FI; it is able, however,

to inhibit the C3 convertase activity (McRae et al., 2005).

1.3.1.3 C4b-binding protein

C4bp is a large (570 kDa), spider-like glycoprotein (Chung et al., 1985; Dahlbäck,

1983; Dahlbäck et al., 1983), present in plasma at a concentration of 200 μg/ml. It

circulates complexed with the vitamin K-dependent protein S. Protein S, bound to

SCR1 of the C4bp �-chain (Härdig et al., 1993; Härdig and Dahlbäck, 1996), does not

display its anticoagulant function (Dahlbäck, 1986). It can, however, direct C4bp to

negatively charged surfaces and apoptotic cells (Schwalbe et al., 1990; Webb et al.,

2002).

C4bp consists of seven identical �-chains (70 kDa) and one �-chain (45 kDa) (Hillarp

and Dahlbäck, 1990). The �- and �-chains are composed of eight and three SCRs,

respectively (Fig. 5). These chains are linked together by disulfide bonds at their most

C-terminal parts (Kask et al., 2002).

1

2

3

4

5

6

7

8

N

�-chain

�-chain

C4b/heparin binding sites

Figure 5. Schematic structure of C4bp (modified from Blom et al., 2004). Binding sites forC4b indicated.


- 17 -

C4bp down-regulates complement activity (Fig. 3), acting as a fluid-phase inhibitor of

the classical and lectin pathway activation steps that involve C4b (Barnum, 1991).

High C4bp concentrations, however, can also force decay of AP C3 convertase (Blom

et al., 2003). N-terminal SCRs 1-3 of C4bp �-chains bind C4b (Kask et al., 2002),

which renders C4b susceptible to FI-mediated cleavage (Fig. 2). In addition, C4bp

prevents the assembly of the CP C3-convertase (C4b2a) and accelerates a natural

decay of the preformed convertase by forcing C2a dissociation from the complex

(Gigli et al., 1979). Heparin can disturb the interaction between C4bp and C4b because

it also binds SCRs 1-3 of C4bp �-chains (Blom et al., 2001; Hessing et al., 1990).

1.3.2 Membrane-bound complement regulators

In addition to fluid phase regulators, cells are equipped with membrane-bound

complement inhibitors that block complement activation at C3 (DAF, MCP, CR1) or

MAC level (CD59) (Morgan and Harris, 1999). Some pathogens, however, use these

membrane-bound regulators for adhesion or invasion, or both, because they are widely

expressed by human cells (Table 3).

1.4 Functions of the complement system

1.4.1 Opsonin-mediated clearance of microbes and host debris

A crucial complement function is to facilitate the phagocytosis of pathogens and the

clean-up of host cell debris. Phagocytic cells are known to bear complement receptors

such as CR1 (CD35), CR3 (CD11b/CD18), and CR4 (CD11c/CD18). CR1 recognizes

the targets coated with C1q, C3b, C4b, and iC3b, whereas the latter two are iC3b-

specific (Klickstein et al., 1988, 1997; Krych et al., 1992; Krych-Goldberg and

Atkinson, 2001; Newman et al., 1984). These opsonins immobilize microbes on the

phagocytic cell surface, which ensures their efficient uptake and destruction.

- 18 -

Reg

ulat

orF

unct

ion

Stru

ctur

eR

egul

ator

yac

tivi

tyP

atho

gens

Ref

eren

ce

MC

P(C

D46

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tor

for

FI-

med

iate

dcl

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f C

3b a

nd C

4b51

-68

kDa

tran

smem

bran

egl

ycop

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in

4 S

CR

dom

ains

tran

smem

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and

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eiss

eria

men

ingi

tidi

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rept

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cus

pyog

enes

Lis

zew

ski

et a

l., 1

991;

Nan

iche

et a

l., 1

993;

Dör

ig e

t al.,

199

3;K

älls

tröm

et a

l., 1

997;

Oka

da e

t al.,

199

5

DA

F(C

D55

)A

ccel

erat

es d

ecay

of

CP

and

AP

C3

conv

erta

ses

Lig

and

for

leuk

ocyt

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tiva

ting

ant

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CD

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70 k

Da

GP

I-an

chor

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rote

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4 S

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corn

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Esc

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ann

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1996

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vans

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Alm

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199

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, 199

3

CR

1(C

D35

)C

ofac

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for

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med

iate

dcl

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f C

3b a

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4b

Acc

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dec

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Pan

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nver

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Cof

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leav

age

to C

3c a

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Imm

une

com

plex

espr

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and

cle

aran

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200

kDa

tran

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bran

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in

30 S

CR

SCR

1-2

SCR

8-9

SCR

15-

16

Pla

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CD

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)In

hibi

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AC

for

mat

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C5b

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18-2

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glyc

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4Y, 4

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61Y

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Esc

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Hel

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hman

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on w

ith

path

ogen

s


- 19 -

Immune complexes, small and soluble antigens derived from antigen-antibody

complexes, are found in the circulation after most infections. C1q binds to immune

complexes or to blebs of apoptotic cells (Korb and Ahearn, 1997; Navratil et al.,

2001). C1q initiates the complement activation on their surfaces and that results in the

deposition of C3b and C4b (Nauta et al., 2002). Apoptotic cells are subsequently

recognized by tissue macrophages and phagocytosed. Ligation of C3b and C4b

deposited on the immune complexes with CR1 on erythrocytes initiates removal of the

complexes from the circulation (Schifferli et al., 1986). Erythrocytes carry this cargo

to the liver and spleen to finally pass it on to macrophages.

1.4.2 Direct lysis of pathogens

Activation of the complement system leads to the generation of MAC on the surface of

target cells, thus causing their death. The complement system by this means plays an

important role in host defense against Gram-negative bacteria. Gram-positive bacteria

are, however, resistant to direct killing mediated by complement. A thick and

impenetrable peptidoglycan layer protects the cytoplasmic membrane of Gram-positive

bacteria from such MAC-induced damage. Although the complement system does not

directly kill Gram-positive bacteria, it still controls the course of Gram-positive

infections by its ability to opsonize and tag the microbes for phagocytic destruction

(Moffitt and Frank, 1994).

1.4.3 Induction of inflammation

Complement cleavage fragments such as C3a, C4a, and C5a are able to induce a local

inflammatory response (Hugli, 1981). Of the three, C5a has the highest inflammation-

inducing activity. These anaphylatoxins act as stimulators and chemoattractants for

those myeloid cells bearing anaphylatoxin-specific receptors. In addition, they act on

blood vessels, increase vascular permeability, and induce endothelial cells for

expression of adhesin molecules (DiScipio et al., 1999; Foreman et al., 1996; Gerard

and Gerard, 1994). This all contributes to a rapid recruitment of phagocytic cells to the

site of inflammation and promotes clearance of pathogens.

1.4.4 Shaping the specific immune response

Complement-derived fragments influence the antibody response of adaptive immunity.

CD19 and CD21 (CR2) are constituents of the B cell co-receptor complex. Binding of


- 20 -

C3d to CD21 triggers CD19-mediated signaling, and this leads to B cell activation

(Dempsey et al., 1996; Dempsey and Fearon, 1996). In addition, the �-chain of C4bp

can activate B cells via the CD40 receptor in a manner similar to that of CD40L

(Brodeur et al., 2003).

1.5 Bacterial mechanisms of complement evasion

Pathogenic microbes have developed a range of strategies to evade complement-

mediated recognition and destruction. To achieve this goal, they have equipped

themselves with surface factors which either directly interfere with the complement

activation or bind host complement regulatory proteins. Microbial complement-

evasion strategies include interference with the initial complement activation, with C3

convertase functions, or with MAC insertion.

1.5.1 Interference with initial complement activation

1.5.1.1 Binding of immunoglobulins via Fc portion

Some microbes interfere with classical pathway activation via binding of the

immunoglobulin Fc portion, e.g., protein G of streptococci or protein A of

Staphylococcus aureus (Björck and Kronvall, 1984; Forsgren and Sjöquist, 1966).

Because the ligation of the Fc receptor on phagocytic cells with the Fc region of target-

bound IgG leads to efficient phagocytosis, blocking of the Fc portion by protein A

inhibits this process (Foster, 2005). In addition, it interferes with C1q binding to IgG,

thereby inhibiting complement activation (Laky et al., 1985).


- 21 -

1.5.1.2 Immunoglobulin and C3/C3b degradation

Several microbes directly or by use of host factors degrade immunoglobulins and

C3/C3b and block the complement activation. Cysteine proteinases, IdeS (IgG-

degrading enzyme of Streptococcus pyogenes) and SpeB (streptococcal pyrogenic

exotoxin B), of group A streptococci remove the Fc portion of bacterium-bound IgG

(von Pawel-Rammingen and Björck, 2003). Porphyromonas gingivalis prtH-encoded

protease degrades both IgG and C3 (Schenkein et al., 1995), whereas S. aureus

degrades these complement-activation initiators indirectly. S. aureus first binds host

plasminogen and converts it to plasmin with the help of staphylokinase. In

consequence, plasmin attached to the bacterial surface cleaves both: C3b and IgG

(Rooijakkers et al., 2005b). Yeast, Candida albicans, proteinases can also degrade C3

and IgG and prevent complement activation (Kaminishi et al., 1995).

1.5.1.3 Binding of C1 inhibitor or C1q inhibition

The binding and utilization of C1 inhibitor (C1INH) occurs for several bacteria.

Enterohemorrhagic E. coli O157:H7 secretes StcE, a protease of C1-INH. StcE recruits

C1-INH to the cell surface and cleaves it to potentiate its ability to inhibit CP (Lathem

et al., 2004). This prevents complement activation and in consequence may restrict an

inflammatory response at the infection site. In addition, Bordetella pertussis, the

etiological agent for whooping cough, recruits C1-INH from human serum to prevent

its own destruction (Marr et al., 2007). Another mechanism to block the CP activation

at C1 level has been reported to take place in helminthic parasites. Paramyosins from

Taenia solium and Schistosoma mansoni bind C1q, block its activity, and prevent

complement activation at its very early stage (Laclette et al., 1992).

1.5.2 Inhibition of C3 convertases

1.5.2.1 Microbial factors directly interfering with C3 convertases

Some pathogens directly interfere with C3 convertase function. Staphylococcal

complement inhibitor (SCIN) is a secreted protein that binds to AP- and CP/LP-C3

convertases, impairing their enzymatic activity. Because such binding stablilizes C3

convertases, it also prevents the formation of new convertases (Rooijakkers et al.,

2005a). Another mechanism is exploited by group A streptococci. SpeB degrades host


- 22 -

serum properdin to inhibit the formation of functional AP C3-convertase and resist

opsonophagocytosis (Tsao et al., 2006). Some microbes also disassemble C3

convertases. Herpes simplex virus (HSV) type 1 expresses the glycoprotein gC-1

which accelerates decay of the pre-formed C3 convertase. In addition, gC-1 binds C3b

and blocks its interaction with properdin, thereby inhibiting formation of AP C3

convertase (Fries et al., 1986; Kostavasili et al., 1997).

1.5.2.2 Microbial factors mimicking FI-cofactors to accelerate decay of the C3

convertases

Some microorganisms mimic the host cofactors for FI-mediated cleavage of C3b and

prevent their own destruction by using a mechanism similar to that exploited by

mammalian cells. Trypanosoma cruzi, a causative agent of Chagas disease, expresses a

glycoprotein gp160 that is functionally and genetically similar to DAF; gp160 is GPI-

anchored, accelerates decay of C3 convertase, and has a coding sequence displaying

�60% similarity to the DAF-encoding gene (Norris et al., 1991). On the other hand,

the amino acid sequence of the vaccinia virus C-control protein (VCP) shows

homology to the C4bp sequence. This accounts for a functional similarity between the

two proteins; SCR-composed VCP binds C4b, prevents formation of CP C3

convertase, and in addition accelerates decay of pre-formed convertases (Kotwal and

Moss, 1988; Kotwal et al., 1990).

1.5.2.3 Microbial factors binding host complement regulators

Pathogens that do not express surface factors that mimick host regulatory proteins

often interact with host complement regulators to protect their own surfaces. Several

microorganisms bind specific fluid-phase regulators, such as FH, FHL-1 and C4bp that

trigger C3 convertase decay and C3b/C4b inactivation.

Pathogens that acquire FH or FHL-1 gain an ability to disassemble AP C3 convertases

and to degrade any C3b deposited on their surfaces. The interaction of FH with the

sialic acid-covered host cells is known to block AP activation (Fearon, 1978). To bind

FH, pathogens such as group B streptococci (Marques et al., 1992) and N .

gonorrhoeae (Ram et al., 1998b) cover their surfaces with sialic acid whereas


- 23 -

trypomastigotes of T. cruzi express an unusual cell-surface trans-sialidase that enables

the parasite to sialylate its surface rapidly (Tomlinson et al., 1994).

Bacterial surface proteins also exist that are able to bind to FH, to FHL-1, or both.

BbCRASP-3 to -5 of Borrelia burgdorferi (Kraiczy et al., 2001), the � protein of

group B streptococcus (Areschoug et al., 2002), Por1A of N. gonorrhoea (Ram et al.,

1998a), or Hic and PspC of Streptococcus pneumoniae (Dave et al., 2001; Janulczyk et

al., 2000) bind FH, whereas BaCRASP-3 of Borrelia afzelii can interact with FHL-1

(Kraiczy et al., 2001). Some microbial proteins bind both FH and FHL-1. These

include M protein and fibronectin-binding protein (Fba) of S. pyogenes (Horstmann et

al., 1988; Kotarsky et al., 2001; Pandiripally et al., 2002) and BbCRASP-1 and

BbCRASP-2 of B. burgdorferi (Kraiczy et al., 2001).

FH-binding sites have been identified for several of these proteins (Fig. 6): BbCRASP-

1, -3 and -5 engage the C terminus; � protein, PspC, and Hic bind to the middle part;

and M protein and Fba interact with the N-terminal region of FH (Blackmore et al.,

1998a; Dave et al., 2004; Duthy et al., 2002; Jarva et al., 2002, 2004; Kotarsky et al.,

1998; Kraiczy et al., 2001; Pandiripally et al., 2003).

Several microbes express surface proteins snatching the host CP/LP regulator, C4bp,

to accelerate decay of CP C3 convertase and degrade C4b deposited on the microbial

surface. These proteins include the M protein of S. pyogenes, Por1A and Por1B of N.

gonorrhoeae, OmpA of E. coli K1, UspA1 and UspA2 of Moraxella catarrhalis, and

FHA of Bordetella pertussis (Berggård et al., 1997; Johnsson et al., 1996; Nordström

Figure 6. Schematic representation of factor H domains interacting with microbial surfaceproteins (modified from Zipfel et al., 2002).


- 24 -

et al., 2004; Prasadarao et al., 2002; Ram et al., 2001). Most of the C4bp-binding

proteins interact with the very N-terminal SCRs of C4bp �-chains, whereas UspA-1

and UspA-2, in addition to N-terminal SCR2, bind C-terminal SCR5 and SCR7 (Fig.

7) (Accardo et al., 1996; Berggård et al., 2001; Nordström et al., 2004; Prasadarao et

al., 2002; Ram et al., 2001).

1.5.3 Interference with MAC formation (terminal pathway)

1.5.3.1 Physical barriers

Gram-positive bacteria are resistant to complement-mediated killing due to the

presence of thick peptidoglycan layer that prevents access of C5b-9 to the cytoplasmic

membrane (Joiner et al., 1983). Also long O-polysaccharide side chains of Salmonella

minnesota interfere with MAC insertion into the outer bacterial membrane. C5b-9 is

generated on the distal parts of LPS molecules which sterically hinder access of MAC

to the outer membrane (Joiner et al., 1986).

Figure 7. Schematic representation of C4bp domains interacting with microbial surfaceproteins (modified from Blom et al., 2004). Binding sites for bacterial factors on C4bp �-chains indicated.

1

2

3

4

5

6

7

8

N

�-chain

M protein

FHA

PorA1

PorA2

OmpA

UspA1

UspA2

�-chain

C4bp domains interacting with microbial proteins


- 25 -

1.5.3.2 Microbial proteins interfering with membrane attack complex

Resistance to the final step of the complement cascade is a prerequisite for the

resistance of Gram-negative bacteria to complement-mediated cytolysis. One example

of protein strongly inhibiting the terminal complement pathway is the E. coli outer

membrane protein TraT. It probably inhibits the formation of C5b-6 complex or causes

its structural alteration to render it nonfunctional (Pramoonjago et al., 1992). Another

example is B. burgdorferi CD59-like protein, which like human membrane-bound

CD59, prevents C9 polymerization and formation of MAC (Pausa et al., 2003).

1.5.3.3 Acquisition of host terminal pathway regulators

To date, few reports describe recruitment of normally membrane-bound terminal

pathway regulators from serum; for instance, E. coli and H. pylori bind CD59 from the

fluid phase to resist MAC-induced lysis (Rautemaa et al., 1998, 2001). On the other

hand, M. catarrhalis UspA2 binds a fluid-phase regulator, vitronectin, to block MAC

insertion into the cell membrane (Attia et al., 2006).

1.5.4 Interference with generation and function of complement-derived

chemoattractants

Gram-positive bacteria, though resistant to MAC-induced cytolysis, inhibit the

terminal pathway to prevent the release of complement chemoattractants such as C5a.

S. aureus, for example, produces a superantigen-related protein SSL7 that binds to C5

and in consequence blocks its cleavage into C5a and C5b (Langley et al., 2005). Group

A and Group B streptococci do not prevent C5 cleavage but inactivate the C5a formed.

Cell wall-anchored C5a peptidase expressed by these bacteria, though unable to cleave

the whole C5, efficiently cleaves C5a (Chmouryguina et al., 1996; Cleary et al., 1992;

Wexler et al., 1985; Wexler and Cleary, 1985). Another strategy to avoid C5a action

has been developed by S. aureus. It secretes CHIPS, an inhibitory protein, that binds to

receptors for C5a and formylated peptides (C5aR and FPR, respectively) on

phagocytic cells. This prevents the phagocytic cells from sensing the early signs of

bacterial infection such as C5a (de Haas et al., 2004).


- 26 -

1.6 Genus Yersinia

Yersinae are Gram-negative rods belonging to the family Enterobacteriaceae. The

genus Yersinia comprises fourteen species. Three of them are human pathogens: Y.

pestis and the enteropathogens Y. pseudotuberculosis and Y. enterocolitica (Brubaker,

1991). All three species harbor a 70-kb virulence plasmid (pYV) essential for immune

evasion and survival in mammalian host (Cornelis et al., 1998) and commonly display

a tropism for lymphoid tissue (Brubaker, 1991). Enteropathogenic yersiniae are motile

at 25°C and non-motile at 37°C due to repression of fliA, the positive regulator of

flagellin streuctural genes. Y. pestis is non-motile regardless of the growth temperature

due to several mutations in falgellar genes (reviewed in (Minnich and Rohde, 2007). In

addition to its difference in motility, Y. pestis causes a distinctive disease and differs in

ecology and epidemiology from the enteropathogenic yersiniae.

1.6.1 Y. pestis

Y. pestis is a causative agent of plague, a systemic infection in mammals. Bubonic

plague is initiated by the bite of an infected flea. As the proventriculus of the flea is

blocked by Y. pestis aggregates, the flea regurgitates the bacterial mass into the bite

site when it attempts to feed (Bacot and Martin, 1914). By means of the flea bite,

bacteria are injected intradermally into the mammalian host, where they begin to

multiply at the site of infection. Via the lymphatic system and bloodstream, bacteria

disseminate to the lymph nodes (Perry and Fetherston, 1997). The bacteria multiply in

the lymph nodes and induce an inflammatory response resulting in swollen and painful

lymph nodes, termed buboes. As the infection proceeds, bacteria enter the

bloodstream, causing septicemia, and infect deeper organs, such as the liver and spleen

(Brubaker, 1991; Perry and Fetherston, 1997). When bacteria reach the lungs, the

disease takes the form of highly infectious pneumonic plague. Individuals with

pneumonic plague transmit the bacteria by spreading infectious droplets. Inhalation of

Y. pestis aerosols results in development of a pneumonic plague and death due to

rapidly progressing sepsis.

Y. pestis harbors, in addition to the pYV, common in enteropathogenic yersiniae, two

extra plasmids pMT1 (pFra) and pPla (pPCP1) (Hu et al., 1998; Lindler et al., 1998).

Ability to survive in the flea gut is provided by pMT1-encoded phospholipase D

(Yersinia murine toxin) while pPla-encoded plasminogen activator, Pla, is required for


- 27 -

the bacteria to penetrate mammalian tissues and disseminate from the site of the

inoculum (Brubaker, 1991; Hinnebusch et al., 2002). Flea-borne transmission requires

a chromosomally located hemin storage locus (hms). Hms-encoded outer membrane

proteins contribute to biofilm formation and blocking of the flea proventriculus

(Hinnebusch et al., 1996).

The three biovars of Y. pestis are differentiated on the basis of their ability to ferment

glycerol and reduce nitrate: Antiqua, Mediaevalis and Orientalis. Historically, each

biovar is associated with one of the three pandemics, the Justinian plague (541-767)

has been assigned to the biovar Antiqua, the Black Death (1346-1850) to Mediaevalis

(Devignant, 1951; Guiyoule et al., 1994), and Biovar Orientalis is responsible for the

most recent plague epidemics (1894-present). This pandemic-biovar relationship has

been recently substantiated by ribotyping (Guiyoule et al., 1994) and by IS1 0 0

insertion element RFLP (restriction fragment length polymorphism) analyses of Y.

pestis strains (Achtman et al., 1999). Y. pestis strains subjected to comparative

sequencing analyses show negligible nucleotide variation, as Y. pestis is a recently

emerged pathogen (Achtman et al., 1999; Adair et al., 2000). However, IS100-based,

multi locus VNTR (variable-number tandem repeat) and single nucleotide

polymorphism (SNP) marker analyses provide the high resolution required for

estimation of genetic relationship of Y. pestis strains (Achtman et al., 1999; Adair et

al., 2000; Touchman et al., 2007).

1.6.2 Enteropathogenic Yersiniae

The enteropathogenic Yersiniae are widespread. Yersiniae infect an array of animal

hosts, including mammals and avian species. Y. pseudotuberculosis and Y.

enterocolitica are usually acquired by ingestion of contaminated food or water (Naktin

and Beavis, 1999). They pass through the acidic content of the stomach and reach the

small intestine. There, they are shuttled across the intestinal epithelium by the M cells

of Peyer’s patches and gain access to subepithelial space (Autenrieth and Firsching,

1996; Marra and Isberg, 1997; Simonet et al., 1990). Subsequently, the bacteria reach

the mesenteric lymph nodes and in some cases spread further to the liver and spleen,

where they multiply and induce an inflammatory response resulting in gastroenteritis

with mesenteric lymphadenitis and terminal ileitis. Y. enterocolitica due to expression

of the heat-stable toxin (Yst) causes more severe and often bloody diarrhea in children

than does Y. pseudotuberculosis (Delor and Cornelis, 1992). Although the disease is


- 28 -

mostly self-limiting and restricted to the gastrointestinal tract, bacteria may

occasionally induce secondary post-infectious sequelae such as arthritis, erythema

nodosum, myocarditis or glomerulonephritis (Baba et al., 1991; Bottone, 1997;

Davenport et al., 1987; Krober et al., 1983; Leino et al., 1980; Takeda et al., 1991;

Tertti et al., 1984).

1.6.2.1 Y. pseudotuberculosis

Both Y. pseudotuberculosis and Y. enterocolitica are soil- and water-borne

enteropathogens causing gastroenteritis. Y. pestis, which causes a distinct disease, and

is an obligate pathogen found solely in arthropod vectors and mammalian hosts, is

paradoxically very closely related to Y. pseudotuberculosis. In fact, population genetic

studies have suggested that Y. pestis evolved from Y. pseudotuberculosis 9,000 to

40,000 years ago (Achtman et al., 1999; Achtman, 2004). Furthermore, Y. pestis and Y.

pseudotuberculosis genome sequences show nearly 97% homology (Chain et al.,

2004). As the LPS O-ag gene cluster of Y. pseudotuberculosis serotype O:1b shows

98.9% identity to the cryptic O-ag gene cluster of Y. pestis, evidence is strong, that the

plague bacterium evolved from Y. pseudotuberculosis serotype O:1b (Skurnik et al.,

2000). Y. pseudotuberculosis is grouped into 21 serotypes based on differences in LPS

O-ag profiles (Skurnik et al., 2000). Based on the distribution of superantigen toxins

(YPMa-c), high-pathogenicity island (HPI) and R-HPI (only the right-hand part of the

HPI present), Y. pseudotuberculosis strains can be further divided into six (genetic)

subgroups (Fukushima et al., 2001) (Table 4). Serotypes of subgroups 1, 3, and 4 are

predominantly found in the Far East (Russia, China, Korea, and Japan); serotypes of

subgroup 2 in the West (Europe, America, Australiasia); and strains belonging to

subgroups 3 and 5 can be isolated throughout the world.

Many of the insecticidal toxin genes occur in Y. pseudotuberculosis, which has, among

Enterobacteriaceae, a unique ability to infect fleas. Interestingly, in Y. pestis many

insecticidal toxin genes are pseudogenes (Hinchliffe et al., 2003; Parkhill et al., 2001)

because the plague bacterium has to persist in the flea gut and needs the living flea for

its life cycle.


- 29 -

Y. pseudotuberculosis

non-pathogenic low-pathogenic pathogenic

subgroups

4 5 1 3 2 6

YPMb+ YPMc+ YPMa+ YPM-

HPI- R-HPI+ HPI+ HPI- HPI+ HPI-

pYV- pYV+ pYV+ pYV+ pYV+ pYV+

Serotypes

O:1b

O:5a

O:5b

O:6

O:7

O:9

O:10

O:11

O:12

O:3 O:1b

O:3

O:5a

O:5b

O:1b

O:2a

O:2b

O:2c

O:3

O:4a

O:4b

O:5a

O:5b

O:6

O:10

O:1a

O:1b

O:1b

O:2a

O:2b

O:2c

O:3

O:4a

O:4b

O:5a

O:5b

O:6

O:7

O:11

O:13

1.6.2.2 Y. enterocolitica

Y. enterocolitica has evolved independently from Y. pseudotuberculosis and Y. pestis

(Wren, 2003). Based on their biochemical heterogeneity, six Y. enterocolitica

biogroups are identifiable (Table 5). Non-pathogenic bacteria lacking the pYV belong

to the biogroup 1A, whereas pathogenic pYV-bearing Y. enterocolitica are divided into

two lineages: low-virulence bacteria including biogroups 2 to 5 (non-lethal in mice)

Table 4. Y. pseudotuberculosis subgroups (modified from Fukushima et al., 2001).


- 30 -

and high-virulence biogroup 1B (lethal in mice) (Bottone, 1997). These biogroups

differ in their geographic distribution; strains belonging to biogroup 1B are found

predominantly in North America, biogroups 2 to 5 strains mainly in Europe and Japan,

and strains belonging to biogroup 1A are distributed throughout the world (Bottone,

1997). These biogroups are further subgrouped into serotypes based on variation in the

LPS O-ag (Table 5).

Y. enterocolitica

avirulent low virulencehigh

virulence

biogroups

1A 2 3 4 5 1B

serotypes

O:5

O:6,30

O:7,8

O:18

O:46

O:9

O:5,27

O:1,2,3

O:5,27

O:3 O:2,3 O:8

O:4

O:13a,b

O:18

O:20

O:21

1.6.3 Virulence determinants of pathogenic Yersiniae

The three pathogenic Yersinia species require a 70-kb virulence plasmid (pYV),

encoding the proteins of the type III secretion system for full virulence (Viboud and

Bliska, 2005). However, pYV, although necessary, alone is not sufficient to cause

disease (Heesemann and Laufs, 1983; Heesemann et al., 1984). Yersinia pathogenesis

additionally involves multiple genes located in the chromosome of the bacterium.

Since Y. enterocolitica and Y. pseudotuberculosis lifestyle differs dramatically from

that of Y. pestis, several plasmid- and chromosome-located genes required for

enteropathogenicity are pseudogenes in the plague bacterium (Tables 6 and 7).

Table 5. Y. enterocolitica biogroups and serotypes (modified from Bottone, 1999).

- 31 -

Fact

or

Fu

nct

ion

Y. en

tY

. pstb

Y. pesti

sR

efer

ence

Ail

(att

achm

ent

and

inva

sion

locu

s)at

tach

men

t to

and

inv

asio

n in

to t

issu

e cu

ltur

e ce

ll

resi

stan

ce t

o co

mpl

emen

t-m

edia

ted

kill

ing

Yes

Yes

No

Yes

Yes

Yes

Bar

tra

et a

l., 2

007;

Bli

ska

and

Fal

kow

, 199

2; K

olod

ziej

ek e

t al.

,20

07;

Mil

ler

and

Fal

kow

, 198

8;Y

ang

et a

l., 1

996

Inv

(inv

asin

)bi

nds �

1-in

tegr

ins

to p

rom

ote

bact

eria

ltr

ansl

ocat

ion

acro

ss i

ntes

tina

l ep

ithe

lium

requ

ired

for

col

oniz

atio

n of

reg

iona

l ly

mph

nod

es

Yes

Yes

Yes

Yes

–

a

–

Isbe

rg a

nd L

eong

, 199

0; M

arra

and

Isbe

rg, 1

997;

Pep

e an

d M

ille

r,19

93;

Sim

onet

et

al.

, 199

6

Psa

(pH

-6 a

ntig

en)

or Myf

(muc

oid

Yer

sinia

fac

tor)

hem

aggl

utin

atio

n of

ery

thro

cyte

s

adhe

sion

to

euka

ryot

ic c

ells

resi

stan

ce t

o ph

agoc

ytos

is

My

f

No

?

b

?

Psa

Yes

Yes ?

Psa

Yes

Yes

Yes

Bic

how

sky-

Slo

mni

cki

and

Ben

-E

frai

m, 1

963;

Hua

ng a

nd L

indl

er,

2004

; Ir

iart

e et

al.

, 199

3; I

riar

tean

d C

orne

lis,

199

5; L

iu e

t al.

,20

06;

Mar

ra a

nd I

sber

g, 1

997;

Yan

g et

al.

, 199

6

Yst

(Yer

sinia

hea

t-st

able

toxi

n)ac

tiva

tes

guan

ylat

e cy

clas

e to

inc

reas

e cy

clic

GM

P c

once

ntra

tion

in

inte

stin

al c

ells

; in

duce

sfl

uid

loss

fro

m t

he c

ells

and

dia

rrhe

a

Yes

––

Del

or e

t al.

, 199

0; D

elor

and

Cor

neli

s, 1

992;

Rob

ins-

Bro

wne

et

al.

, 197

9

Sod

(sup

erox

ide

dism

utas

e)de

toxi

fica

tion

of

reac

tive

oxy

gen

spec

ies,

resi

stan

ce t

o ba

cter

icid

al a

ctiv

ity

of p

hago

cyti

cce

lls

requ

ired

for

ful

l vi

rule

nce

Yes

Yes

? ?

? No

Rog

genk

amp

et a

l., 1

997;

Seb

bane

et

al.

, 200

6

YP

M(Y

. p

seudotu

ber

culo

sis-

deri

ved

mit

ogen

)

supe

rant

igen

ic t

oxin

tha

t st

imul

ates

pro

life

rati

on o

fpo

lycl

onal

T l

ymph

ocyt

es–

Yes

–A

be e

t al.

, 199

3; M

iyos

hi-

Aki

yam

a et

al.

, 199

3

Tab

le 6

. O

verv

iew

of

the

mos

t im

port

ant

chro

mos

omal

ly-e

ncod

ed v

irul

ence

fac

tors

of

path

ogen

ic Y

ersi

nia

e.

- 32 -

LP

S(l

ipop

olys

acch

arid

e)O

-an

tigen

effe

ctiv

e co

loni

zati

on o

f ho

st t

issu

es i

n ea

rly

stag

eof

inf

ecti

on

resi

stan

ce t

o co

mpl

emen

t-m

edia

ted

kill

ing

Core

inva

sion

of

deep

er t

issu

es

resi

stan

ce t

o ca

tion

ic a

ntim

icro

bial

pep

tide

s

resi

stan

ce t

o co

mpl

emen

t-m

edia

ted

kill

ing

Lip

idA

resi

stan

ce t

o ca

tion

ic a

ntim

icro

bial

pep

tide

s

deac

ylat

ed l

ipid

A a

t 37

ºC:

r

educ

ed b

iolo

gica

l ac

tivi

ty

req

uire

d fo

r th

e sy

stem

ic s

prea

d

Yes

Y

es/N

o c

Yes

Yes

Yes

/No

Yes

(21º

C <

37º

C)

Yes

Yes ?

Yes

No ? ? ?

Yes

(21º

C <

37º

C)

Yes

Yes ?

– – ?

Yes ?

Yes

(21º

C >

37º

C)

Yes

Yes

Yes

Ani

sim

ov e

t al.

, 200

5;B

engo

eche

a et

al.

, 200

3, 2

004;

Bru

bake

r, 1

991;

Kaw

ahar

a et

al.

,20

02;

Kni

rel

et a

l., 2

005;

Mec

sas

et a

l., 2

001;

Mon

tmin

y et

al.

,20

06;

Por

at e

t al.

, 199

5; P

rior

et

al.

, 200

1; R

ebei

l et

al.

, 200

4;S

kurn

ik e

t al.

, 199

9, 2

000;

Wac

hter

and

Bra

de, 1

989

HP

I – e

nco

ded

:(h

igh-

path

ogen

icit

y is

land

)

Y

bt

(yer

sini

abac

tin)

hm

s l

ocu

s(h

emin

sto

rage

)

HP

I pr

esen

t in

hig

hly

path

ogen

ic Y

ersi

nia

e

side

roph

ore

that

cap

ture

s ir

on m

olec

ules

bou

nd t

oeu

kary

otic

pro

tein

s

biof

ilm

for

mat

ion

in t

he f

lea

and

bloc

king

of

flea

prov

entr

icul

us

biog

roup

1B

Yes –

se

roty

pe O

:1 d

Yes

No

all

thre

ebi

ovar

s

Yes

Yes

De

Alm

eida

et

al.

, 199

3; E

rick

son

et a

l., 2

006;

Fuk

ushi

ma

et a

l.,

2001

; H

aag

et a

l., 1

993;

Hin

nebu

sch

et a

l., 1

996;

Per

ry e

t

al.

, 199

9; R

akin

et

al.

, 200

1

a la

ck o

f ex

pres

sion

, b to

be

dete

rmin

ed, c

diff

eren

t se

roty

pes,

d fo

r de

tail

s se

e T

able

4

- 33 -

Pla

smid

/Fact

or

Fu

nct

ion

Y. en

tY

. pstb

Y. pesti

sR

efer

ence

pY

VY

esY

esY

es

Yop

s(Y

ersi

nia

out

er p

rote

ins)

Yop

T

cyst

ein

prot

ease

wit

h an

tiph

agoc

ytic

act

ivit

y; i

nact

ivat

esR

ho G

TP

ases

(R

hoA

, Rac

, Cdc

42)

by r

emov

al o

f li

pid

mod

ific

atio

ns a

nd b

y di

srup

tion

of

acti

n fi

lam

ents

Yop

E

anti

phag

ocyt

ic a

ctiv

ity

due

to i

nact

ivat

ing

GA

P a

ctiv

ity

tow

ards

Rho

GT

Pas

es;

disr

upts

the

act

in c

ytos

kele

ton

Yop

O o

r Y

pk

A (

Yer

sinia

pro

tein

kin

ase

A)

anti

phag

ocyt

ic a

ctiv

ity;

aut

opho

phor

ylat

ing

seri

ne-

thre

onin

e ki

nase

tha

t ta

rget

s G�

q fa

mil

y of

G p

rote

ins,

ther

eby

inhi

biti

ng G�

q-m

edia

ted

sign

alin

g pa

thw

ays

Yop

H

anti

phag

ocyt

ic p

rote

in t

yros

ine

phos

ohat

ase;

tar

gets

p130

Cas

, foc

al a

dhes

ion

kina

se (

FA

K),

pax

illi

n, a

nd F

yn-

bind

ing

prot

ein

to d

isru

pt f

ocal

adh

esio

ns;

redu

ces �

1in

tegr

in p

athw

ay a

ctiv

ated

by

inte

ract

ion

wit

h i

nvas

in;

bloc

ks F

c-m

edia

ted

resp

irat

ory

burs

t of

pha

gocy

tic

cell

s;su

ppre

sses

T-

and

B-c

ell

acti

vati

on

Yes

Yes

Yop

O

Yes

Yes

Yes

Yes

Yp

kA

Yes

Yes

Yes

Yes

Yp

kA

Yes

Yes

Bla

ck a

nd B

lisk

a, 1

997;

Bla

ck e

t

al.

, 199

8; B

lack

and

Bli

ska,

200

0;B

lisk

a an

d B

lack

, 199

5; B

olan

dan

d C

orne

lis,

199

8; C

abel

los

et

al.

, 199

2; C

orne

lis

et a

l., 1

987;

Den

ecke

r et

al.

, 200

1;D

ukuz

umur

emyi

et

al.

, 200

0;E

rfur

th e

t al.

, 200

4; E

vdok

imov

et

al.

, 200

2; G

alyo

v et

al.

, 199

3;G

rosd

ent

et a

l., 2

002;

Ham

id e

t

al.

, 199

9; I

riar

te a

nd C

orne

lis,

1998

; Ju

ris

et a

l., 2

000;

McD

onal

d et

al.

, 200

3; M

ills

et

al.

, 199

7; M

onac

k et

al.

, 199

7;N

avar

ro e

t al.

, 200

7; P

alm

er e

t al.

,19

98, 1

999;

Per

sson

et

al.

, 199

7,19

99;

Ros

qvis

t et

al.

, 199

1;R

uckd

esch

el e

t al.

, 199

6, 1

997,

1998

, 200

1; S

ches

ser

et a

l., 1

998;

Sha

o et

al.

, 200

3; S

krzy

pek

et a

l.,

2003

; S

tral

ey a

nd B

owm

er, 1

986;

Trü

lzsc

h et

al.

, 200

5; V

ibou

d an

dB

lisk

a, 2

001;

Yao

et

al.

, 199

9

Tab

le 7

. O

verv

iew

of

the

mos

t im

port

ant

plas

mid

-enc

oded

vir

ulen

ce f

acto

rs o

f pa

thog

enic

Yer

sinia

e.

- 34 -

Yop

M

a le

ucin

e-ri

ch p

rote

in o

f un

know

n pr

ecis

e ce

llul

ar f

unct

ion;

traf

fics

to

the

nucl

eus

via

a ve

sicl

e-as

soci

ated

pat

hway

;fo

rms

a co

mpl

ex w

ith

two

cyto

plas

mic

kin

ases

RS

K1

and

PR

K2

ther

eby

acti

vati

ng t

hem

Yop

P/Y

op

J

an a

nti-

infl

amm

ator

y cy

stei

n pr

otea

se;

indu

ces

apop

tosi

s in

phag

ocyt

ic c

ells

, inh

ibit

s M

AP

K s

igna

ling

pat

hway

s vi

abi

ndin

g to

mul

tipl

e m

embe

rs o

f th

e M

AP

K s

uper

fam

ily,

e.g.

MK

Ks

and

IKK�

; bl

ocks

NF

-�B

act

ivat

ion

thro

ugh

inhi

biti

on o

f I�

B d

egra

dati

on, i

nhib

its

TN

F-�

and

IL

-8pr

oduc

tion

, sup

pres

ses

CD

8 T

cel

l re

spon

se

Yes

Yop

P

Yes

Yes

Yop

P

Yes

Yes

Yop

J

Yes

Yad

A(Y

ersi

nia

adh

esin

A)

adhe

sion

to

host

cel

ls, e

pith

elia

l br

ush

bord

er, a

nd m

ucus

entr

y in

to h

ost

cell

s vi

a fi

bron

ecti

n-bo

und

inte

ract

ion

wit

h�

1-in

terg

rin

rece

ptor

s

hem

aggl

utin

atio

n of

ery

thro

cyte

s

auto

aggl

utin

atio

n

seru

m r

esis

tanc

e

indu

ctio

n of

IL

-8 p

rodu

ctio

n by

epi

thel

ial

cell

s

bind

ing

to E

CM

pot

eins

:

c

olla

gen

type

s I,

II,

IV

fib

rone

ctin

lam

inin

Yes

No

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

–

a

– – – – – – – –

Bal

liga

nd e

t al.

, 198

5; B

lisk

a et

al.

, 199

3; B

ruba

ker,

196

7; C

hina

et a

l., 1

993;

Eit

el a

nd D

ersc

h,20

02;

Eit

el e

t al.

, 200

5; E

l T

ahir

et a

l., 2

000;

Em

ödy

et a

l., 1

989;

Flü

gel

et a

l., 1

994;

Hee

sem

ann

and

Grü

ter,

198

7; H

eise

and

Der

sch,

200

6; K

appe

rud

et a

l.,

1987

; M

antl

e et

al.

, 198

9;M

arti

nez,

198

9; M

ota

et a

l., 2

005;

Pae

rreg

aard

et

al.

, 199

1a, 1

991b

;P

ilz

et a

l., 1

992;

Rog

genk

amp

et

al.

, 199

6; R

osqv

ist

et a

l., 1

988;

Ruc

kdes

chel

et

al.

, 199

6; S

chm

idet

al.

, 200

4; S

chul

ze-K

oops

et

al.

,19

92, 1

993;

Sku

rnik

et

al.

, 198

4;S

kurn

ik a

nd W

olf-

Wat

z, 1

989;

Tam

m e

t al.

, 199

3; T

ertt

i et

al.

,19

92;

Yan

g an

d Is

berg

, 199

3

- 35 -

Lcr

V o

r V

-an

tigen

anti

-inf

lam

mat

ory,

sup

pres

ses

TN

F-�

and

IF

N-�

prod

ucti

on a

nd i

nduc

es I

L-1

0 re

leas

e, i

nhib

its

neut

roph

ilch

emot

axis

Yes

Yes

Yes

Nak

ajim

a et

al.

, 199

5; S

ing

et a

l.,

2002

; W

elko

s et

al.

, 199

8

pP

la (

pP

CP

1)

––

Yes

Pla

(pla

smin

ogen

act

ivat

or)

conv

erts

pla

smin

ogen

int

o pl

asm

in a

nd i

nact

ivat

es t

he m

ain

plas

min

inh

ibit

or (

anti

-pro

teas

e �

2-an

tipl

asm

in)

med

iate

s ad

hesi

on t

o ex

trac

ellu

lar

mat

rix

degr

ades

fib

rin

clot

s an

d ex

trac

ellu

lar

mat

rix

clea

ves

com

plem

ent

fact

or C

3

redu

ces

chem

oatt

ract

ion

of t

he i

nfla

mm

ator

y ce

lls

requ

ired

for

the

dev

elop

men

t of

pri

mar

y pn

eum

onic

pla

gue

enha

nces

bac

teri

al a

dhes

ion

to e

pith

elia

l ce

ll l

ines

– – – – – – –

– – – – – – –

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Kie

nle

et a

l., 1

992;

Kuk

kone

n et

al.

, 200

1; L

ähte

enm

aki

et a

l.,

1998

; L

athe

m e

t al.

, 200

7;S

odei

nde

et a

l., 1

992

pM

T1

(p

Fra

)–

–Y

es

Ym

t(Y

ersi

nia

mur

ine

toxi

n) p

hosp

holi

pase

inv

olve

d in

the

col

oniz

atio

n an

d su

rviv

al o

fY

. pes

tis

in t

he m

idgu

t of

the

fle

a–

–Y

esH

inne

busc

h et

al.

, 200

2

F1 c

ap

sule

anti

phag

ocyt

ic, r

educ

es b

acte

rial

bin

ding

to

phag

ocyt

icce

lls

––

Yes

Du

et a

l., 2

002

a la

ck o

f ex

pres

sion


- 36 -

1.6.4 Y. enterocolitica surface factors shaping serum resistance phenotype

1.6.4.1 YadA

YadA, formerly known as Yop1 or P1, forms lollipop-like structures (approx. 30 nm or

23 nm in length in Y. enterocolitica serotypes O:3 and O:8, respectively) on the surface

of enteropathogenic Yersiniae (Bölin et al., 1982, 1985; Hoiczyk et al., 2000). YadA is

the most important virulence factor of Y. enterocolitica in the initial intestinal stage of

the infection (Pepe et al., 1995). The role of YadA in Y. pseudotuberculosis infection

remains unclear, however, as the yadA mutant is as virulent as the wild type Y.

pseudotuberculosis strain (Bölin and Wolf-Watz, 1984). In Y. pestis, YadA is silenced

as a result of a single nucleotide deletion in the yadA gene (Rosqvist et al., 1988;

Skurnik and Wolf-Watz, 1989). The Y. pestis lifestyle differs dramatically from that of

Y. enterocolitica and Y. pseudotuberculosis. The YadA required for adhesion to gut

tissue appears to be redundant for the plague bacterium and therefore is represented by

the pseudogene.

Expression of the yadA gene is thermodependent and under the control of the virF/lcrF

gene which encodes for a transcriptional activator driving YadA expression at 37°C

but not below 30°C (Lambert de Rouvroit et al., 1992; Skurnik and Toivanen, 1992).

YadA is a homotrimeric 200- to 240-kDa outer membrane protein with a monomer

size of about 44 kDa (Mack et al., 1994; Nummelin et al., 2004; Skurnik et al., 1984).

YadA belongs to the family of trimeric autotransporters and consists of an internal

passenger domain and a C-terminal translocation unit (Fig. 8) (Cotter et al., 2005;

Hoiczyk et al., 2000; Roggenkamp et al., 2003; Skurnik and Wolf-Watz, 1989; Surana

et al., 2004). The YadA passenger domain consists of N-terminal head, neck, and

coiled-coil stalk domains and is followed by a C-terminal translocation and membrane-

anchoring unit (Hoiczyk et al., 2000). A detailed description of the YadA structural

units is given below for the Y. enterocolitica serotype O:3. The membrane anchor

represents a 12-stranded �-barrel closed by a stalk segment of four heptamers (residues

368-395) (Koretke et al., 2006). The left-handed coiled-coil segment continues into a

right-handed coiled-coil (residues 214-367) stalk built of 15-residue repeats

(pentadecads) (Hoiczyk et al., 2000). The number of these degenerate repeats is strain-

specific. The right-handed part of the stalk of Y. enterocolitica serotype O:3 has been

predicted to consist of one exceptional 19-residue unit following the left-handed

segment, and nine pentadecads (Koretke et al., 2006). The right-handed part of the


- 37 -

stalk is connected to the neck region (residues 192-213) that comprises a safety pin-

like element of each monomer (Nummelin et al., 2004) and makes the transition from

the rod-like stalk to the globular head. The head domain, about 5 nm long, is a tight

cylindrical trimeric structure formed by left-handed �-rolls (Nummelin et al., 2004).

The export of autotransporters through the inner membrane, involving the Sec

machinery, is initiated by the signal peptide. Once through the inner membrane, the

signal sequence is cleaved off, and the translocation domain is inserted into the outer

membrane, forming a �-barrel structure (Cotter et al., 2005). This structure constitutes

a pore in the outer membrane through which the YadA passenger domain extrudes.

The exact mechanism by which the passenger domain is translocated through the pore

remains, however, poorly understood. Once all three YadA subunits have been

translocated, the protein folds completely and achieves its native conformation. As

YadA has no cysteines (Skurnik and Wolf-Watz, 1989), its trimerization depends

strongly on ionic or hydrophobic interaction, or both, between the polypeptide chains.

Head

Neck

Stalk

Anchor

Autoagglutination

Binding to: collagen

laminin

fibronectin

neutrophils

epithelial cells

complement resistance

translocation

oligomerization

complement resistance

PAS

SE

NGER

DOMAIN

TU

Figure 8. Schematic representation of YadA domains and their functions (modified fromKoretke et al., 2006); TU, translocation unit.


- 38 -

YadA appears on bacterial surface within minutes after a temperature-shift to 37°C

(Bölin et al., 1982). Because YadA molecules on neighboring bacteria interact with

each other in a zipper-like fashion, involving the head and neck domains, bacteria tend

to autoagglutinate (Balligand et al., 1985; Hoiczyk et al., 2000).

YadA is unusual in a number of its functions (Table 7). It binds to mucin (Paerregaard

et al., 1991a) to facilitate bacterial colonization of the intestine, and it mediates the

binding of bacteria to �1-integrins on the host cells via a fibronectin bridge (Eitel and

Dersch, 2002). The latter activity, specific for Y. pseudotuberculosis YadA, which is

bearing a unique N-terminal sequence (Heise and Dersch, 2006), may stimulate

phagocytic activity of the M cells to transport Yersiniae across the intestinal

epithelium. Y. enterocolitica YadA binds fibronectin (Schulze-Koops et al., 1993;

Tertti et al., 1992) but does not mediate invasion of human epithelial cells (Heesemann

and Grüter, 1987; Schmid et al., 2004). In addition to fibronectin, YadA also binds to

other extracellular matrix proteins such as collagen types I, II, III, IV, V, and XI

(Emödy et al., 1989; Schulze-Koops et al., 1992), and laminin types 1 and 2 (Flügel et

al., 1994).

YadA also mediates adherence to cultured human epithelial cells (Bliska et al., 1993;

Heesemann and Grüter, 1987), rabbit intestinal tissue (Paerregaard et al., 1991b),

guinea pig erythrocytes (Kapperud and Lassen, 1983; Kapperud et al., 1985), human

intestinal submucosa (Skurnik et al., 1994), and phagocytic cells such as neutrophils

(Roggenkamp et al., 1996) and macrophages (Mota et al., 2005). YadA binding to

macrophages has been shown to anchor the bacteria so that the type III secretion

needle can reach the host cell membrane, and the effector proteins can be translocated

to the cytoplasm of the host cell (Mota et al., 2005).

1.6.4.1.2 YadA-mediated serum resistance

One of the multiple functions of YadA is bactericidal serum resistance. This functional

characteristic is shared by several members of the oligomeric coiled-coil adhesin (Oca)

family (Table 8). YadA protects bacteria against bactericidal action of serum as

transposon insertion yadA mutants prove susceptible to human serum, and, in addition,

YadA expressed in E. coli K12 confers serum resistance upon this normally serum-

sensitive bacterium (Balligand et al., 1985; Martinez, 1989). Attempts to identify the

mechanism of YadA-mediated complement resistance implicated YadA in the


- 39 -

inhibition of complement activation at both C3b and C9 levels (China et al., 1993; Pilz

et al., 1992). Bacteria expressing YadA were also shown more rapidly degrade

surface-bound C3b to iC3b, and accordingly, display higher resistance to opsonin-

mediated phagocytosis (Grosdent et al., 2002; Pilz et al., 1992). It has been proposed

that YadA may silence the complement activation and reduce C3b-opsonization by

coating the bacterial surface with FH (China et al., 1993; Roggenkamp et al., 1996).

No direct binding of FH to YadA, however, has been demonstrated.

Protein Organism Serum resistance

mechanism

Reference

Omp100(outer membrane protein 100)

Actinobacillus

actinomyctemcomitans

FH binding Asakawa et al.,2003

DsrA(ducreyi serum resistance A)

Haemophilus ducreyi C4bp bindingvitronectin binding

Abdullah et al.,2005

UspA1 and UspA2(ubiquitous surface protein A)

M. catarrhalis C4bp bindingvitronectin binding

Nordström et

al., 2004; Attiaet al., 2006

Eib(E. coli Ig binding)

E. coli unknown Sandt and Hill,2000

Hsf(Haemophilus surface fibrils)

Haemophilus

influenzae

vitronectin binding Hallström et al.,2006

A study aimed at identification of YadA segments contributing to serum resistance

argued that this function could not be ascribed to a single YadA domain (Roggenkamp

et al., 2003). Both the head domain and the neck region appeared to be required for the

YadA-mediated complement resistance (Roggenkamp et al., 2003). It has been

proposed, however, that regions within the stalk and the translocation domain function

as serum-resistance determinants (Ackermann et al., 2008; Roggenkamp et al., 2003).

Table 8. Members of the YadA family conferring serum resistance.


- 40 -

1.6.4.2 Ail

Ail is a 17-kDa outer membrane protein encoded by the chromosomally located ail

gene (Miller and Falkow, 1988; Miller et al., 1990). The presence of this gene has

been associated exclusively with the pathogenic Y. enterocolitica species (Miller et al.,

1989). Transcription of the ail gene is regulated by the temperature and the growth

phase of the bacteria. In log-phase bacteria, the ail transcript is detectable at 22°C,

28°C, and 37°C, whereas in the stationary-phase bacteria, Ail is expressed exclusively

at 37°C (Pierson and Falkow, 1993). In addition, Ail expression is reduced when

bacteria grow under limited oxygen conditions (Pederson and Pierson, 1995).

Ail belongs to a family of proteins predicted to form eight outer membrane-spanning

amphipatic �-strands and four short extracellular loops (Fig. 9) (Beer and Miller, 1992;

Stoorvogel et al., 1991; Vogt and Schulz, 1999). These loops appear to be masked to

some extent by the full-length LPS (Pierson, 1994).

Ail mediates attachment to, and subsequent invasion of yersinia into eukaryotic cells

(Miller and Falkow, 1988) but the exact mechanism of Ail-mediated invasion is

unknown. The role for Ail in vivo also remains to be determined, since ail-defective

bacteria cross the gastrointestinal epithelium in a similar fashion as do the wild type

bacteria (Wachtel and Miller, 1995). Nevertheless, Ail is indeed produced by bacteria

in vivo during the first two days of infection (Wachtel and Miller, 1995).

1.6.4.2.1 Ail-mediated serum resistance

The family of outer membrane proteins structurally related to Ail includes E. coli and

Enterobacter cloacae OmpX, and Salmonella typhimurium PagC and Rck (Beer and

Miller, 1992; Heffernan et al., 1992; Mecsas et al., 1995; Pulkkinen and Miller, 1991;

Stoorvogel et al., 1991). Only Rck, however, shares functional homology with Ail;

namely its resistance to complement (Heffernan et al., 1994). Ail has been implicated

in resistance to complement-mediated killing based on the following observations: (i)

pathogenic Y. enterocolitica ail-defective mutant bacteria became sensitive to serum

killing and the serum-resistance phenotype of the strain was restored by

complementation by a wild-type copy of the ail gene, (ii) introducing ail into

laboratory E. coli strains and non-pathogenic strains of Y. enterocolitica resulted in


- 41 -

increased resistance to killing by human serum (Bliska and Falkow, 1992; Pierson and

Falkow, 1993). The serum resistance-conferring regions of Y. enterocolitica serotype

O:8 Ail have been mapped to the C-terminal end of loop 2 and the N-terminal end of

loop 3 (Miller et al., 2001).

1.6.4.3 LPS

LPS is the major component of the outer membrane of Y. enterocolitica; it has three

main structural components: lipid A, the core (inner and outer), and O-ag. Unlike in

many other Gram-negative bacteria, in Y. enterocolitica serotypes O:3 and O:9, the O-

ag and the OC are linked to the inner core (IC), forming a branched structure on the

bacterial surface (Fig. 10) (Skurnik et al., 1995; Skurnik and Bengoechea, 2003).

1.6.4.3.1 O-antigen

O-ags of Y. enterocolitica serotypes O:3 and O:9 are homopolymers of 6-deoxy-L-

altrose and N-formylperosamine, respectively (Caroff et al., 1984; Hoffman et al.,

1980).

Figure 9. Schematic representation of the E. coli OmpX structure (modified fromFernandez et al., 2002); loops 1-4 (L1-4).


- 42 -

Homopolymeric O-ags are fully synthesized at the cytoplasmic face of the inner

membrane, on the membrane-bound carrier undecaprenyl phosphate (Und-P). Und-P

accepts the first sugar moiety from nucleotide diphosphate (NDP)-activated sugar

precursors that function as sugar donors. This reaction and the sequential transfer of

sugar moieties to the growing polysaccharide polymer is carried out by specific

glycosyltransferases. The full-length O-ag polymer is translocated to the periplasm via

ATP-driven transporter system formed of the members of ABC transporter family.

Subsequently the polymer is ligated by the O-ag ligase to pre-formed lipid A-core and

translocated to the outer membrane (Raetz and Whitfield, 2002).

The Y. enterocolitica serotype O:3 O-ag gene cluster is composed of two operons (Fig.

11), both essential for O-ag synthesis and both transcribed from tandem promoters

(Al-Hendy et al., 1991a, 1992; Zhang et al., 1993). These operons are likely to encode:

(i) enzymes involved in the biosynthesis of the NDP-activated sugar precursor, dTDP-

6-deoxy-L-altrose (wbbS, wbbV and wbbW), (ii) two glycosyltransferases to initiate the

O-ag synthesis and to extend the homopolymer (wbbT and wbbU), and (iii) ABC

transporter system (wzt and wzm) (Skurnik, 1999; Zhang et al., 1993). The genomic

location of the O-ag gene cluster is unknown. The organization and characterization of

the O-ag gene cluster of serotype O:9 is presented in Study II.

O-antigen

Outer core

Inner core

Lipid A

Figure 10. A schematic drawing of LPS of Y. enterocolitica serotype O:3 (modified fromSkurnik et al., 1995).


- 43 -

O-ag expression is regulated by the temperature and the growth phase. Below 30°C, O-

ag expression by bacteria in the stationary phase (or near the start of the stationary

phase of growth) is maximal. Bacteria in the exponential phase, however, express O-

ag constitutively and regardless of the growth temperature (Lahtinen et al., 2003).

Y. enterocolitica serotype O:3 O-ag serves as a receptor for bacteriophage �YeO3-12

(Al-Hendy et al., 1991a). In addition, it plays a role in the virulence of Y .

enterocolitica and appears to be required for effective colonization of host tissues

during the very first hours of infection (Al-Hendy et al., 1992; Skurnik et al., 1999).

1.6.4.3.2 Outer core

OC gene clusters of Y. enterocolitica serotypes O:3 and O:9 are very closely related

and are located between the hemH and gsk genes. Interestingly, this locus in Yersinia is

usually occupied by heteropolymeric O-ag gene clusters (Skurnik et al., 1995;

Skurnik and Bengoechea, 2003). OC of Y. enterocolitica serotype O:3 is, however,

thought to be an ancestral O-unit which serves as a receptor for bacteriophage �R1-37

and enterocoliticin (Skurnik et al., 1995; Strauch et al., 2003).

Hexasaccharide OC of Y. enterocolitica serotype O:3 forms a branch consisting of two

glucoses, two N-acetyl-D-galactosamines, galactose, and 2-acetamido-2,6-dideoxy-D-

xylo-4-hexulose (Duda, 2007; Skurnik, 1999). The latter sugar links the OC to the IC.

OC biosynthesis begins at the cytoplasmic face of the inner membrane on the Und-P.

The transfer of sugar residues—from NDP-sugar precursors—to the growing

hexasaccharide is carried out by glycosyltransferases. Completed hexasaccharide is

flipped to the periplasmic space, and subsequently ligated to the lipidA-inner core

structure (Skurnik, 1999).

wbbS wbbT wbbU wzm wzt wbbV wbbW wbbX

P3,4P1,2

Figure 11. O-ag gene cluster of Y. enterocolitica serotype O:3, schematic drawing(modified from Skurnik and Bengoechea, 2003). Individual genes are drawn as arrows butnot to scale. Colour of each arrow indicates gene function; grey, biosynthetic enzyme;white, glycosyltransferase; and black, polysaccharide transporter. P1,2 and P3, 4, tandempromoters.


- 44 -

The Y. enterocolitica serotype O:3 OC gene cluster consists of nine genes presumably

encoding: (i) enzymes involved in the biosynthesis of the NDP-activated sugar

precursors (wbcP and gne), (ii) six glycosyltransferases (wbcK, wbcL, wbcM, wbcN,

wbcO, and wbcQ), and (iii) a flippase (wzx) (Fig. 12) (Skurnik et al., 1995; Skurnik

and Bengoechea, 2003).

OC is required for the full virulence of Y. enterocolitica serotype O:3, for colonization

of the deeper organs, and for prolonged persistence of the bacteria in the Peyer’s

patches (Skurnik et al., 1999). OC provides also resistance against antimicrobial

peptides and that suggests its role in resistance to innate host defense mechanisms

(Skurnik et al., 1999).

1.6.4.3.3 LPS and serum resistance

Although a role for O-ag of Y. enterocolitica in the inhibition of AP activation is

suggested based on the studies using the guinea pig serum, studies using human serum

do not provide evidence for involvement of O-ag and OC in serum resistance (Skurnik

et al., 1999; Wachter and Brade, 1989).

Figure 12. OC gene cluster of Y. enterocolitica serotype O:3, schematic drawing (modifiedfrom Skurnik and Bengoechea, 2003). Individual genes are drawn as arrows but not to scale.The color of each arrow indicates gene function; gray, biosynthetic enzyme; white,glycosyltransferase; and black, flippase. Dashed-line arrows represent genes flanking theOC cluster.

AIMS OF THE STUDY

- 45 -

2. AIMS OF THE STUDY

This thesis work was undertaken to increase our understanding of molecular details of

the interactions between the Y. enterocolitica surface factors and serum complement

components. The specific aims of this study were:

1. to determine the role of Y. enterocolitica surface factors YadA, Ail, and LPS in

bacterial resistance against complement-mediated killing.

2. to investigate whether Y. enterocolitica utilizes the AP inhibitor FH and the CP

inhibitor C4bp, to resist the complement system.

3. to identify and map the regions of the bacterial complement resistance factors

interacting with the complement regulators.

MATERIALS AND METHODS

- 46 -

3. MATERIALS AND METHODS

The materials and methods of this study are listed below, with detailed descriptions in

the original publications, which are here referred to by Roman numerals.

Bacterial strains DescriptionSource or

reference

Used

in

Y. enterocolitica O:3

6471/76 (YeO3) Serotype O:3, patient isolate, wild type Skurnik, 1984 I, III,V

31761 Serotype O:3, fecal isolate HUSLAB, Helsinki,Finland

V

49008 Serotype O:3, blood isolate HUSLAB, Helsinki,Finland

V


V

YeO3-Ail �ail::Km-GenBlock, Kmr This study I, III

YeO3-Ail-R Spontaneous rough derivative of YeO3-Ail,Kmr

This study I, III

YeO3-Ail-OCR Spontaneous OC mutant derivative of YeO3-Ail-R, Kmr

This study I, III

YeO3-trs-11 �(wzx-wbcL)::Km-GenBlock, Kmr, derivativeof YeO3

Skurnik et al., 1995 I, III

YeO3-trs-11R Spontaneous rough derivative of YeO3-trs11,Kmr


YeO3-OC �(wzx-wbcQ), derivative of YeO3 This study I, III

YeO3-OCR Spontaneous rough derivative of YeO3-OC This study I, III

YeO3-Ail-OC �ail::Km-GenBlock, Kmr, derivative of YeO3-OC

This study I, III

YeO3-R2 Spontaneous rough derivative of YeO3 Al-Hendy et al., 1992 I, III

YeO3-O28 �yadA::Km-GenBlock, Kmr, derivative ofYeO3

This study I, III,V

YeO3-O28-R Spontaneous rough derivative of YeO3-O28,Kmr

This study I, III,V

YeO3-O28-OCR Spontaneous OC mutant derivative of YeO3-O28-R1, Kmr

This study I, V

YeO3-O28-OC �(wzx-wbcQ) derivative of YeO3-O28, Kmr This study I, III,V

6471/76-c(YeO3-c)

Virulence plasmid cured derivative of YeO3 Skurnik, 1984 I, III,IV,V

YeO3-R1 Spontaneous rough derivative of YeO3-c Al-Hendy et al., 1992 I, III,V

Table 9. Bacterial strains used in this study


- 47 -

YeO3-c-OC �(wzx-wbcQ) derivative of YeO3-c This study I, III,V

YeO3-c-OCR Spontaneous rough derivative of YeO3-c-OC This study I, III,V

YeO3-c-trs8 �(wzx-wbcL)::Km-GenBlock, Kmr, derivativeof YeO3-c


YeO3-c-trs8R Spontaneous rough derivative of YeO3-c-trs8,Kmr


YeO3-c-Ail �ail::Km-GenBlock, Kmr, derivative ofYeO3-c

This study I, III,IV,V

YeO3-c-Ail-OC Spontaneous OC mutant derivative ofYeO3-c-Ail, Kmr

This study I, III,V

YeO3-c-Ail-R Spontaneous rough derivative of YeO3-c-Ail,Kmr

This study I, III,V

YeO3-c-Ail-OCR Spontaneous OC mutant derivative ofYeO3-c-Ail-R, Kmr

This study I, III,IV,V


8081 Serotype O:8, wild type Portnoy et al., 1981 V

YeO8-116 �yadA::Km-GenBlock, derivative of 8081,Kmr

This work V

90937(ATCC 23715)

Serotype O:8, quality assessment strain,Labquality, Helsinki, Finland, pYV-negative

HUSLAB, Helsinki,Finland

V


90936 Serotype O:9, quality assessment strain HUSLAB, Helsinki,Finland

V


V

Ruokola/71 Patient isolate Skurnik, 1984 II

Ruokola/71-c Spontaneous virulence plasmid curedderivative of Ruokola/71

Skurnik, 1984 II

YeO9-R1 �per::Km-GenBlock, rough (O-ag negative),Kmr derivative of Ruokola/71

This work II

YeO9-OC �(wzx-wbcL)::Km-GenBlock, OC negative,Kmr derivative of Ruokola/71

This work II

YeO9-OCP Phage �R1-37 resistant spontaneous OC-low-expressing derivative of Ruokola/71

This work II

E. coli

C600 thi thr leuB tonA lacY supE Appleyard, 1954 I, V

DH10B F- mcrA �(mrr-hsd RMS-mcrBC),�80lacZ�M15 �lacX74, deoR, recA1 endA1

ara�139�(ara, leu)7697 galU, galK �- rpsL

nupG �- tonA

Life Technologies IV

HB101/pRK2013 Triparental conjugation helper strain, Kmr Figurski and Helinski,1979

I, IV,V

JM103 Sequencing host strain Messing et al., 1981 III,V


- 48 -

JM109 reaA1 �lac-pro endA1 gyrA96 thi-1 hsdR17

supE44 relA1 F’ traD36 proAB+ lacIqZ�M15

Yanisch-Perron et al.,1985

IV,V

S17-1 recA derivative of E. coli 294 (F- thi pro hsdR)carrying a modified derivative of IncP�plasmid pRP4 (Aps Tcs Kms) integrated in thechromosome, Tpr

Simon et al., 1983 V

S17-1�pir thi pro hsdR- hsdM+ recA::RP4-2-Tc::Mu-Km::Tn7, Strr (�pir)

De Lorenzo andTimmis, 1994

I, II

SY327�pir �(lac pro) argE (Am) rif nalA recA56 (�pir) Miller and Mekalanos,1988

I, II


- 49 -

Plasmids DescriptionSource or

reference

Used

in

pAil-8 A pBR322 clone carrying a 8-kb insert of YeO3-ccontaining the ail gene, Ampr

This study I

pAil-8.1 PvuII deletion derivative of pAil-8, Ampr This study I

pAil-8.2 EcoRV-NruI deletion derivative of pAil-8.1, Ampr This study I

pBR322 Cloning vector, Ampr Tetr Bolivar et

al., 1977I

pJM703.1 Suicide vector, contains R6K origin of replication andRP4 Mob region; must be replicated in (�pir) hosts,Ampr

Miller andMekalanos,1988

I

pL2.1 pBR322 with the Ptac promoter, Ampr Viitanen et

al., 1990III,V

pRV1 Suicide vector, derivative of pJM703.1, Clmr Skurnik et

al., 1995I

pRV7 A pBR322 clone carrying a 12.4-kb insert of YeO3-ccovering the OC gene cluster, Ampr

Skurnik et

al., 1995I

pRV19-GB Suicide vector to inactivate OC gene cluster, �(wzx-wbcL)::Km-GenBlock, Clmr Kmr

Skurnik et

al., 1995II

pRV37 Intermediate to construct OC deletion suicide vector;pBR322 carries a HindIII fragment of pRV7 fromwhich the OC gene cluster is deleted by PCR, Ampr

This study I

pRV38 2-kb HindIII fragment of pRV37 cloned into pRV1(O:3 OC gene cluster suicide vector), Clmr

This study I

pRV42 Suicide vector constructed to inactivate the ail gene;Km-GenBlock inserted between ail flanking fragmentsand cloned into pRV1, Kmr, Clmr

This study I

pTM100 Mobilizable vector, pACYC184-oriT of RK2, Clmr Michiels andCornelis,1991

I,IV,V

pTM100-ail ail gene cloned in a 1711 bp PCR fragment(nucleotides 1490-3201**) into EcoRV site ofpTM100, Clmr

This study IV,V

pTM100-ail1 bp 2459-2461 CAC substituted for GCA inpTM100-ail; expresses Ail with His65Alasubstitution (loop2)

This study IV

pTM100-ail2 bp 2465-2470 GATCTT substituted forGGAGGT in pTM100-ail; expresses very lowlevels of Ail with Asp67Gly and Leu68Glysubstitutions (loop2)

This study IV

pTM100-ail3 bp 2465-2470 GATCTT substituted forGCACGA in pTM100-ail; expresses very lowlevels of Ail with Asp67Ala and Leu68Argsubstitutions (loop2)

This study IV

pTM100-ail4 bp 2564-2566 TCA substituted for GAT inpTM100-ail; expresses Ail with Ser100substituted for Asp (loop3)

This study IV

pUC-4K Origin of the Km-GenBlock cassette, Ampr, Kmr Pharmacia I, V

Table 10. Plasmids used in this study


- 50 -

pYMS3221x yadA cloned as 4.4-kb XbaI-PvuI fragment betweenEcoRV-XbaI sites of pTM100

This study V

pYMS3223 Km-GenBlock inserted in the ClaI site of pYMS3221x This study V

pYMS 4450 promoterless yadA cloned into pL2.1; Ampr Skurnik et

al., 1994III,V

pYMS 4505 yadA gene of YeO3 cloned as a 1735 bp PCR fragment(nucleotides 478-2213*) into BamHI site of pTM100;Clmr

El Tahir et

al., 2000I

pYMS4505:�NECK bp 1194-1271 deletion of the yadA gene in pYMS 4505;expresses YadA with 26 aa deletion (aa 193-218)

This study IV

pYMS4505:A bp 1257-1301 deletion of the yadA gene in pYMS 4505;expresses YadA with 15 aa deletion (aa 214-228)

This study IV

pYMS4505:S bp 1281-1325 deletion of the yadA gene in pYMS 4505;expresses YadA with 15 aa deletion (aa 222-236)

This study IV

pYMS4505:�#1 bp 1311-1355 deletion of the yadA gene in pYMS 4505;expresses YadA with 16 aa deletion (aa 231-246)

This study IV

pYMS4505:B bp 1347-1391 deletion of the yadA gene in pYMS 4505;expresses YadA with 15 aa deletion (aa 244-258)

This study IV


This study IV

pYMS4505:C bp 1359-1403 deletion of the yadA gene in pYMS4505; does not express YadA due to a frame shiftmutation introduced during the construction

This study IV

pYMS4505:D bp 1371-1415 deletion of the yadA gene in pYMS4505; expresses YadA with 15 aa deletion (aa252-266)

This study IV

pYMS4505:E bp 1383-1427 deletion of the yadA gene in pYMS4505; expresses YadA with 15 aa deletion (aa256-270)

This study IV

pYMS4505:�#3+6 bp 1398-1448 deletion of the yadA gene in pYMS4505; expresses YadA with 17 aa deletion (aa261-277)

This study IV

pYMS4505:F bp 1392-1436 deletion of the yadA gene in pYMS4505; expresses YadA with 15 aa deletion (aa259-273)

This study IV

pYMS4505:�#3 bp 1404-1448 deletion of the yadA gene in pYMS4505; expresses YadA with 15 aa deletion (aa263-277)

This study IV

pYMS4505:H bp 1416-1460 deletion of the yadA gene in pYMS4505; expresses YadA with 15 aa deletion (aa267-281)

This study IV

pYMS4505:I bp 1428-1472 deletion of the yadA gene in pYMS4505; expresses YadA with 15 aa deletion (aa271-285)

This study IV


This study IV

pYMS4505:J bp 1482-1571 deletion of the yadA gene in pYMS4505; expresses YadA with 30 aa deletion (aa289-318)

This study IV


This study IV


- 51 -

pYMS4505:�#5-6 bp 1494-1583 deletion of the yadA gene in pYMS4505; expresses YadA with 30 aa deletion (aa293-322)

This study IV

pYMS4505:K bp 1506-1595 deletion of the yadA gene in pYMS 4505;expresses YadA with 30 aa deletion (aa 297-326)

This study IV

pYMS4505:L bp 1518-1607 deletion of the yadA gene in pYMS 4505;expresses YadA with 30 aa deletion (aa 301-330)

This study IV


This study IV

pYMS4505:�#6+3 bp 1539-1586 deletion of the yadA gene in pYMS 4505;expresses YadA with 16 aa deletion (aa 308-323)

This study IV

pYMS4505:M bp 1572-1616 deletion of the yadA gene in pYMS 4505;expresses YadA with 15 aa deletion (aa 319-333)

This study IV

pYMS4505:�#7-9 bp 1584-1706 deletion of the yadA gene in pYMS4505; expresses YadA with 41 aa deletion (aa323-363)

This study IV

pYMS4505:N bp 1617-1661 deletion of the yadA gene in pYMS4505; expresses YadA with 15 aa deletion (aa334-348)

This study IV

pYMS4505:O bp 1662-1706 deletion of the yadA gene in pYMS4505; expresses YadA with 15 aa deletion (aa349-363)

This study IV

pYMS4505:P bp 1707-1727 deletion of the yadA gene in pYMS4505; expresses YadA with 7 aa deletion (aa 364-370)

This study IV

pYMS4505:�1-3 bp 1707-1772 deletion of the yadA gene in pYMS4505; does not express surface-located YadA, 22aa deletion (aa 364-385)

This study IV

pYMS4505:Q bp 1728-1748 deletion of the yadA gene in pYMS4505; expresses YadA with 7 aa deletion (aa 371-377)

This study IV

pYMS4505:R bp 1749-1769 deletion of the yadA gene in pYMS4505; expresses YadA with 7 aa deletion (aa 378-384)

This study IV

pYMS 4506 �yadA::Km-GenBlock derivative of pYMS4505; theinternal 1.3-kb NsiI fragment of yadA gene replacedwith Km-GenBlock; Clmr, Kmr

This study I

pYMS 4509 The �yadA::Km-GenBlock fragment cloned intoEcoRI site of pJM703.1; Ampr, Kmr

This study I

pYMS 4515 Suicide vector to inactivate the yadA gene; the cat

gene of pTM100 cloned into the PstI site ofpYMS4509; Clmr, Kmr

This study I

pYV8081 Virulence plasmid of 8081 Portnoy et

al., 1981V

* in all yadA mutants, nucleotide positions refer to the numbering of the 2551 bp sequence containingthe yadA-gene of Y. enterocolitica O:3 under Acc. no. X13882.** in all ail mutants, nucleotide positions refer to the numbering of the 3865 bp sequence containing theail-gene of Y. enterocolitica O:3 under Acc. no. AJ605740


- 52 -

Antibody name

or cat. no Description

Source or

reference

Used

in

Primary

antibodies

2A9 Mouse monoclonal antibody against YadA ofY. enterocolitica O:3, epitope within aa 193-244

Skurnik et al., 1994 III,IV

2G12 Mouse monoclonal antibody against YadA ofY. enterocolitica O:3, epitope within aa 193-214

Skurnik et al., 1994 III,IV

3G12 Mouse monoclonal antibody against YadA ofY. enterocolitica O:3, epitope within aa 236-263

Skurnik et al., 1994 I,III,IV

Tom A6 Mouse monoclonal antibody against O-ag ofY. enterocolitica O:3

Pekkola-Heino et

al., 1987I

2B5 Mouse monoclonal antibody against OC ofY. enterocolitica O:3

Pekkola-Heino et

al., 1987I

755 Mouse monoclonal antibody against �-chain ofhuman C3

Vogel et al., 1997 I

C2456 Mouse monoclonal antibody against collagen type I SIGMA IV

A0062 Rabbit antiserum against human C3c DAKO III

A0063 Rabbit antiserum against human C3d DAKO III

A312 Goat antiserum against human FH QUIDEL III,IV

PC026 Sheep antiserum against human C4bp The Binding Site,Birmingham, UK

V

A0065 Rabbit antiserum against human C4c DAKO V

Secondary

antibodies

P0260 Peroxidase-conjugated rabbit anti-mouseimmunoglobulins

DAKO I,IV

P217 Peroxidase-conjugated swine anti-rabbitimmunoglobulins

DAKO III,IV,V

P449 Peroxidase-conjugated rabbit anti-goatimmunoglobulins

DAKO III,IV

713-035-147 Peroxidase-conjugated donkey anti-sheepimmunoglobulins

JacksonImmunoResearchLaboratories

V

Table 11. Antibodies used in this study


- 53 -

Bacteriophages

and bacteriocins Description

Source or

reference

Used

in

Bacteriophages

�YeO3-12 O-antigen specific phage of Y. enterocolitica O:3 Al-Hendy et al.,1991a

I

�R1-37 Outer core specific phage of Y. enterocolitica O:3 Skurnik et al.,1995

I,II,III

�R8-01 Infects O-ag and OC-negative Y. enterocolitica O:3bacteria

This study III

Enterocoliticin Outer core-specific bacteriocin Strauch et al.,2001, 2003

I

Purified protein DescriptionSource or

reference

Used

in

FH Human Calbiochem(341274)

III,IV

FI Human Calbiochem(341280)

III,IV,V

Collagen type I Human SIGMA(C7774)

IV

C3b Human; generated from C3 by factor B, factor D inthe presence of Mg2+

This study andKoistinen et al.,1989

III,IV

C4b Human Calbiochem(204897)

V

SCR 1-5 Human, recombinant FH construct produced inthe baculovirus expression system

This study andKühn and Zipfel,1995

III,IV

SCR 1-6 Human, recombinant FH construct produced in

the baculovirus expression system


III,IV




III,IV




III,IV




III,IV




III,IV

Table 12. Bacteriophages and bacteriocins used in this study

Table 13. Purified proteins used in this study


- 54 -

MethodsUsed

in

Site-directed mutagenesis I, IV

PCR I, II, IV

Cycloserine enrichment I

Cloning I, IV

Extraction of genomic or plasmid DNA or both I, II, IV, V

Screening of recombinant plasmids by size IV

Conjugation I, II, IV, V

Preparation of electrocompetent cells and electroporation I, IV, V

Construction of suicide vectors to eliminate the ail and yadA genes I

Isolation of spontaneous mutants using bacteriophages I, II

DOC-PAGE and silver staining I, II

Collagen affinity blotting IV

Tx-114 or �-octylglucoside isolation of outer membrane proteins III, V

Bacterial outer membrane preparation I

Lipopolysaccharide isolation I, II

Radiolabeling of proteins using Iodine (125I) III, V

Human serum pool preparation I, II, III, IV, V

Enzyme-linked immunosorbent assay III

SDS-PAGE and Immunoblotting I, III, IV, V

Cofactor assay for C3b or C4b inactivation III, IV, V

Direct binding of radiolabeled proteins to Yersinia III, V

Serum bactericidal assay I, II, IV

Serum adsorption assay V

Binding of purified FH, FH recombinant constructs, or C4bp to bacteria III, IV, V

Ligand blotting analysis of FH or C4bp binding III, V

Bacteriophage sensitivity assay III

Modeling of the coiled-coil structure of the YadA mutants IV

Statistical methods II, III, IV

Table 14. Methods used in this study

RESULTS AND DISCUSSION

- 55 -

4. RESULTS AND DISCUSSION

4.1 Combined action of Y. enterocolitica outer membrane proteins and LPS

in resistance to complement-mediated lysis

The complement system contributes to innate immune responses by opsonizing and

killing microorganisms and by augmenting the inflammatory response. Escape from

innate defense mechanisms appears as a prominent evasion strategy of Y .

enterocolitica. By avoiding the activation of host innate defense mechanisms, Y.

enterocolitica, like many pathogenic bacteria, is able to resist the bactericidal activity

of complement. Several bacterial surface factors, studied individually, have been

implicated in the complement resistance. These include YadA, Ail, and LPS O-ag and

OC (Balligand et al., 1985; Bliska and Falkow, 1992; Martinez, 1989; Pierson and

Falkow, 1993; Skurnik et al., 1999; Wachter and Brade, 1989). Studies on the role of

LPS in serum resistance have, however, produced conflicting results (Skurnik et al.,

1999; Wachter and Brade, 1989).

To study the contribution of these four factors, we constructed 23 mutant strains of Y.

enterocolitica O:3 expressing different combinations of YadA, Ail, O-ag, and OC (I).

Survival of bacteria, in the exponential or stationary phase of growth, was tested by

incubating the bacteria in 70% normal human serum (NHS) (CP, LP, and AP

functional) or EGTA-Mg-treated serum (only AP functional). The reaction was

stopped after 0.5 or 2 hours’ incubation at 37°C, the samples were plated, and the

viable counts of the bacteria were determined (I). The results obtained correlated with

previous observations pointing to involvement of YadA and Ail in resistance to

CP/AP-mediated killing. In addition, the importance of the combination of the factors

in the outer membrane into providing optimal protection against serum killing, was

evident.

In general, bacteria growing exponentially were more complement-resistant than

bacteria grown to the stationary phase (Figs 4 and 5 in I). YadA, as the most potent

single-serum resistance factor, provided bacteria with long-term protection against

complement-mediated lysis (Fig. 2 in I). Ail, although crucial for bacterial defense

against serum killing, conferred short-term resistance and delayed the killing of

bacteria. The protective effect of Ail could, however, be seen when YadA-lacking


- 56 -

bacteria were deprived of O-ag and OC (Fig. 3 in I). LPS is predicted to cover 75%

of the bacterial surface (Raetz and Whitfield, 2002). It may thus sterically block small-

sized Ail in the outer membrane and, in consequence, interfere with the Ail-exerted

functions: serum resistance and, as shown earlier, attachment to/invasion of

mammalian cells (Pierson, 1994). Since also the binding of bacteriophages to their

receptors was blocked when these were located beneth the LPS “coat” [(Kawaoka et

al., 1983; Skurnik et al., 1995) and III], it is apparent that LPS can mask the bacterial

surface (Fig. 13).

Although remains unknown, how bacteria modulate LPS expression in vivo, in vitro O-

ag and OC expression is downregulated at 37°C (Al-Hendy et al., 1991a, b;

Muszynski, 2004). Thus, one can assume that bacteria, when needed, are able to

downregulate the expression of LPS in vivo to uncover factors crucial for certain steps

of an infection. This hypothesis appears to be quite applicable in the case of Y.

enterocolitica O:3 LPS, which provided no protection against complement-mediated

killing (Fig. 3 in I). Moreover, bacteria lacking either O-ag or OC displayed enhanced

resistance to CP/LP or AP killing, respectively. Interestingly, this dependence was

apparent only when the three factors YadA, Ail, and either O-ag or OC were expressed

(Fig. 2 in I). Thus, it appears that serum resistance factors, to be fully functional, must

be presented in the membrane in the correct context.

Figure 13. Schematic model of Y. enterocolitica surface illustrating serum resistancefactors.

YadA

Ail

O-ag

OC


- 57 -

In contrast to serotype O:3, LPS appears to participate in serum resistance in serotype

O:9. The importance of long O-ag chains in resistance against complement lysis has

been demonstrated also for other pathogens (Burns and Hull, 1998; Grossman et al.,

1987; Joiner et al., 1986). In Y. enterocolitica O:9, however, LPS plays an accessory

role in serum resistance by strengthening the YadA-mediated protection; OC appears

to contribute to protection in the early exposure to serum, whereas O-ag contributes to

prolonged bacterial survival (Fig. 4 in II).

In general, within a population of the wild type bacteria of both serotypes there existed

serum-sensitive and serum-resistant fractions. One could speculate that the surviving

bacteria are hidden within aggregates typically formed by the YadA-expressing

bacteria. In addition, heterogeneity of the bacterial population may result from

differential expression of bacterial-surface factors. It is known that LPS molecules

isolated from any smooth Y. enterocolitica strain show an extensive variation in the

length of their O-ag chains. Moreover, some of these LPS molecules do not contain

attached O-ag chains (Skurnik, 2004). Thus, one could envision bacterial cells

equipped with fewer or shorter O-ag chains or both as being more resistant to

complement due to the greater exposure of YadA and Ail.

4.2 Y. enterocolitica and acquisition of complement regulators

Complement activation, being potentially deleterious for the host tissues, is tightly

regulated. Fluid-phase complement regulators are, however, often sequestered by

pathogens to prevent complement activation on their surfaces (Berggård et al., 1997;

Hellwage et al., 2001; Horstmann et al., 1988; Johnsson et al., 1996; Meri et al., 2002;

Nordström et al., 2004; Prasadarao et al., 2002; Ram et al., 1998b, 2001). We thus

aimed to verify whether binding of complement regulators such as FH and C4bp

would also be a mechanism of the complement resistance of Y. enterocolitica.

4.2.1 Acquisition of FH

First, we wanted to examine the mechanism by which Y. enterocolitica evades AP-

mediated killing. Although Y. enterocolitica binds FH from serum (China et al., 1993;

Roggenkamp et al., 1996), no direct binding of purified FH to bacteria has been

demonstrated (Roggenkamp et al., 2003). We thus aimed to re-examine whether Y.

enterocolitica acquires FH to prevent AP activation on its surface.


- 58 -

Bacteria, incubated with human serum in the test tube or in microtiter wells were then

washed, and deposited serum FH was analyzed by immunoblotting or ELISA (Fig. 1 in

III). In both techniques, goat antiserum that recognizes human FH served for the

detection of bound FH. As both methods confirmed the earlier observation that Y.

enterocolitica acquires serum FH (Fig. 1 in III), we wanted to examine whether Y.

enterocolitica involves other serum proteins to mediate the interaction with FH.

Bacteria were thus incubated with purified FH, washed, and subjected to SDS-PAGE

and immunoblotting. The analysis, using goat antiserum against human FH, revealed

that Y. enterocolitica O:3 bound purified FH directly (Fig. 2 in III). Since earlier

attempts to demonstrate a direct binding of purified/125I-labeled to Y. enterocolitica

had failed (Roggenkamp et al., 2003), we also re-assayed the binding experiment. We

did not succeed, however, in showing the 125I-FH binding to Y. enterocolitica, either

(III). We thus examined the effect of buffers used in the binding assay and found that

gelatin used to block non-specific binding was likely to inhibit the 125I-FH-Y.

enterocolitica interaction (III). Gelatin is a denatured form of collagen and greatly

inhibits YadA-mediated collagen binding (Emödy et al., 1989). One could thus suggest

that FH-Y. enterocolitica interaction, like that of collagen-Y. enterocolitica, involves

YadA and thus can be inhibited by gelatin.

The specificity of FH binding to Y. enterocolitica was further determined. Bacteria

were incubated with purified FH in the presence of bovine serum albumin (BSA). The

binding of FH was affected only by the highest BSA concentration tested, suggesting

that the binding of FH to Y. enterocolitica is specific (III).

We next aimed to study whether FH bound to the Y. enterocolitica surface remained in

a functional form, by testing the functional activity of Y. enterocolitica-bound FH in a

cofactor assay (Fig. 2 in III). Bacteria pre-incubated with purified FH were incubated

further with FI and C3b. The degree of C3b cleavage was assayed by subjecting the

supernatants to SDS-PAGE/immunoblotting with a mixture of anti-C3c and anti-C3d

antibodies (Fig. 2 in III). Y. enterocolitica-bound FH acted as a cofactor for FI-

mediated cleavage of C3b, as the �’-chain of C3b became cleaved into 67 kDa, 43

kDa, and 41 kDa fragments. Thus, FH deposited on the Y. enterocolitica surface

retained its regulatory function.


- 59 -

4.2.2 Acquisition of C4bp

As Y. enterocolitica resists the CP/LP-mediated killing (Fig. 2 in I), we wanted to

examine whether one mechanism underlying this resistance is acquisition of C4bp. A

serum adsorption assay was used to test whether Y. enterocolitica binds serum C4bp.

Bacteria were incubated in 5% heat-inactivated serum at 37°C; bacteria-bound serum

proteins were then eluted and subjected to SDS-PAGE/immunoblotting with sheep

antiserum against human C4bp (Fig. 3 in V). Because the immunoblotting results

revealed the presence of C4bp in the eluted fractions, we next studied whether Y.

enterocolitica binds C4bp independent of other serum proteins. 125I-labeled C4bp was

incubated with Y. enterocolitica serotypes O:3, O:8, and O:9. The bacteria that bound

radiolabeled C4bp were centrifuged through 20% sucrose, and the binding was

quantified with a gamma counter (Fig. 1 in V). All three Y. enterocolitica serotypes

directly bound C4bp. The specificity of C4bp binding underwent further study in a

direct binding assay. Bacteria were incubated with 125I-C4bp in the presence of

unlabeled C4bp or BSA. The binding of 125I-C4bp was inhibited in a concentration-

dependent manner by unlabeled C4bp, whereas BSA did not affect 125I-C4bp binding.

In conclusion, we found that the binding of C4bp to Y. enterocolitica to be specific

(Fig. 7 in V).

To examine the functional activity of Y. enterocolitica-acquired C4bp, a cofactor assay

was performed. Bacteria pre-incubated with C4bp were subsequently washed and

incubated with C4b and FI. The C4bp-cofactor activity was verified by

immunodetection of C4b cleavage products in the collected supernatant by use of

immunoblotting with rabbit anti-human C4c antiserum (Fig. 2 in V). The results

demonstrated that C4bp bound to Y. enterocolitica maintained its cofactor activity and

participated in C4b inactivation. In conclusion, we found C4bp to bind to Y.

enterocolitica in a way that does not interfere with its regulatory function.


- 60 -

4.3 Identification of FH and C4bp receptors on Y. enterocolitica

Given the Y. enterocolitica - FH and Y. enterocolitica - C4bp interaction, we wanted to

identify the potential receptors for these complement regulators on the surface of Y.

enterocolitica. To this end, we used a set of mutant strains of Y. enterocolitica O:3

expressing different combinations of YadA, Ail, O-ag, and OC, which had served to

identify those bacterial factors that confer serum resistance (I). The ability of the

mutant strains to bind serum FH or C4bp was tested by ELISA/immunoblotting or by

direct binding assay, respectively (Fig. 1 in III and Fig. 4 in V). In general, FH/C4bp

binding was dependent on the presence of those factors that confer complement

resistance, YadA and Ail. Importantly, the FH- and C4bp-binding pattern correlated

well with the serum sensitivities of the strains; the strains of intermediate and resistant

phenotype bound the regulators, whereas the sensitive ones failed to (I, III, V, Fig. 14).

In general, serum-sensitive strains bound negligible amounts of FH and C4bp (Fig.

14). Strains belonging to this group did not express YadA, the most potent single

serum resistance factor. It thus became evident that YadA expression is crucial for

C4bp/FH binding (Fig. 14). Interestingly, although several serum-sensitive strains

expressed Ail—shown earlier to confer serum resistance—Ail’s ability to bind

C4bp/FH was inhibited in the presence of O-ag or OC (YeO3-O28, YeO3-c, YeO3-

O28-OC, YeO3-c-OC, and YeO3-O28-R, Fig. 14). The distal parts of LPS lacked the

ability to bind either of the regulators, and in consequence did not protect the bacteria

against serum killing (YeO3-c-Ail, YeO3-c-Ail-OC, and YeO3-c-Ail-R).

All the strains expressing YadA were able to bind both FH and C4bp (Fig. 14). These

strains displayed intermediate (YeO3, YeO3-Ail, YeO3-Ail-OC, YeO3-Ail-OCR,

YeO3-Ail-R, and YeO3-OCR) or resistant phenotypes (YeO3-OC, YeO3-R2, YeO3-

trs11, and YeO3-trs11R). The only two strains that lacked YadA but were able to bind

FH and C4bp expressed Ail in the absence of both O-ag and OC (YeO3-c-OCR and

YeO3-c-trs8R, Fig. 14). Thus, Ail must be very well exposed in the outer membrane to

be able to bind complement regulators.


- 61 -

Interestingly, the ability of YadA-expressing strains to bind FH was greatly enhanced

in the absence of O-ag (YeO3-R2, YeO3-trs11R, YeO3-OCR, YeO3-Ail-OCR, and

YeO3-AilR, Fig. 14). This suggests that the lack of O-ag might favor FH binding to

YadA by exposing its regions crucial for the binding. Lack of OC in the O-ag- YadA-

expressing strain did not greatly affect bacterial ability to bind FH (YeO3-OC). YeO3-

trs11, however, shown to express less O-ag than did YeO3-OC, bound more FH than

did the wild type (I and Fig. 1 in III).

Figure 14. Y. enterocolitica strains expressing different combinations of YadA, Ail, O-ag,and OC. Serum resistance vs FH/C4bp binding phenotype: sensitive, <1% viable bacteria;intermediate 1-20% viable bacteria; resistant, >20% viable bacteria after 120 minincubation in NHS. Strains marked with an asterisk were tested only for the ability to bindFH. See Fig. 13 for symbols.


- 62 -

4.3.1 Characterization of YadA- Ail- mediated interactions with FH and C4bp

4.3.1.1 YadA and FH

These findings warranted further studies on the YadA- and Ail-mediated interactions

with FH and C4bp (III-V). YadA was initially identified as an FH receptor based on an

affinity-blotting experiment using whole serum as the FH source (China et al., 1993).

To show a direct interaction between FH and YadA, we re-assayed the affinity-

blotting experiment. YadA was extracted from E. coli with Triton X-114, and the

binding of purified FH to the extracted protein was tested in affinity blotting with goat

antiserum against anti-human FH. We observed the binding of purified FH to YadA,

thereby confirming that the interaction between YadA and FH is direct (Fig. 3 in III).

To assess the nature of the YadA-FH interaction, we incubated YadA-expressing

bacteria with purified FH in the presence of salt (50-650 mM) or heparin (0-1000

μg/ml). As salt did not greatly affect the binding of FH to YadA, the YadA-FH

interaction is likely to be hydrophobic in nature (Fig. 5 in III). Heparin had some effect

on FH-binding to YadA only at the highest concentrations used (Fig. 5 in III). This

suggests that the YadA-FH interaction does not depend primarily on SCR 7, SCR 13,

or SCR 20, which have been shown to be heparin-specific (Blackmore et al., 1996;

Blackmore et al., 1998b; Pangburn et al., 1991).

To characterize regions of FH involved in the YadA-FH interaction in more detail, we

incubated YadA-expressing bacteria with truncated recombinant constructs together

representing the entire FH. Binding of FH was detected by immunoblotting with goat

antiserum against human FH. All FH fragments bound to YadA-expressing bacteria,

suggesting that the interaction with YadA engages SCRs of the entire polypeptide

chain of FH (Fig. 15 and Fig. 4A in III). This finding confirmed the heparin inhibition

results. Since the SCR 1-5 construct contains the domain responsible for the regulatory

functions of FH, i.e., its cofactor and decay-accelerating activities (Gordon et al.,

1995; Kühn et al., 1995; Kühn and Zipfel, 1996), we next examined whether FH

bound to YadA remained functional. Despite the fact that YadA-FH interaction

involves SCRs 1-5, YadA-bound FH retained its regulatory function. Many pathogens

like B. burgdorferi, N. gonorrhoeae, and C. albicans target C-terminal SCRs of FH

(Hellwage et al., 2001; Meri et al., 2002; Ram et al., 1998b), S. pneumoniae targets its


- 63 -

middle portion (Jarva et al., 2002), and S. pyogenes and B. burgdorferi target its N-

terminal region (Kotarsky et al., 1998; Kraiczy et al., 2004; Pandiripally et al., 2003).

Binding to the SCRs of the entire FH, however, has not been reported with any other

microbe and thus appears to be unique for YadA.

Since we showed that Y. enterocolitica-bound FH maintains its function, we wanted to

study whether YadA-expressing bacteria use FH to promote FI-mediated cleavage of

C3b (Fig. 2 in III). The cofactor assay results demonstrated that YadA-bound FH was

fully functional. Our C3b deposition experiments, however, did not support this

observation (Fig. 6 in I). The deposition findings not only failed to correlate with

resistance phenotypes of the strains, but also pointed to O-ag as a factor preventing the

deposition of C3b (Fig. 6 in I). Interestingly, no difference between the wild type and

the YadA-negative strains in 125I-C3 deposition from 10% EGTA-Mg serum was

observed by Pilz and co-workers either (Pilz et al., 1992). In contrast to this

observation, Tertti and co-workers reported that pYV+ bacteria displayed less serum

C3b deposited that did pYV- bacteria (Tertti et al., 1987). This difference between

strains only appeard, however, with NHS concentration no higher than 5% (Tertti et

al., 1987). China and co-workers showed, however, reduced C3b deposition, from

10% EGTA-Mg serum, on bacteria expressing YadA (China et al., 1993). As our data

showed no reduction in C3b deposition by YadA-positive strains, one could speculate

that the concentration of serum used in the assay was too high. We cannot at present

explain these discrepancies.

4.3.1.1.1 Mapping of YadA regions involved in FH-binding

As we found that O-ag blocks to some extent the binding of FH to YadA, we wanted to

examine whether O-ag blocks the binding of mAb to YadA in an analogous fashion

(III). We used the mAbs recognizing epitopes within the neck and the very N-terminal

Figure 15. Schematic representation of FH domains interacting with YadA ofY. enterocolitica serotype O:3; lanes indicate recombinant fragments used.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20N C


- 64 -

region of the YadA stalk (2A9, 3G12, and 2G12). The access of these mAbs to their

epitopes was not blocked, however, by the presence of O-ag. This suggested that YadA

regions interacting with FH are located closer to the bacterial outer membrane than are

the mAbs epitopes. The indication that regions of the YadA stalk may be involved in

FH binding and the fact that the YadA stalk, but not the head domain, mediates serum

resistance (Roggenkamp et al., 2003) warranted further studies on characterization of

YadA regions involved in both serum resistance and FH binding (IV). To this end, we

constructed 28 mutants (Fig. 16) having short internal deletions within the neck and

stalk of YadA, based on the proposed coiled-coil structure of the trimeric stalk

(Koretke et al., 2006). Plasmids carrying the mutated yadA gene were mobilized to the

plasmid-cured Y. enterocolitica O:3 host, YeO3-c. Expression of the trimerized and

surface-exposed YadA was detectable in all the mutants, except for the one carrying

deletion �364-385 (Fig. 1 in IV). This finding was consistent with the earlier finding

showing that this region, overlapping the junction between the right-handed and left-

handed parts of the stalk, is crucial for YadA translocation across the outer membrane

(Roggenkamp et al., 2003). Since all our stalk mutants bound collagen type I, the

deletions introduced did not affect the head and neck domains involved in collagen

binding (Fig. 1 in IV). Modeling analysis suggests that most deletions do not disturb

the structure of the stalk, whereas in certain mutant proteins the local periodicity and

thus degree of supercoiling are affected (IV).

The mutants were shown to be useful for the localization of epitopes for the three

mAbs specific for YadA: 3G12-15, 2A9, and 2G12. The epitopes were mapped to aa

236-263, 193-244, and 193-214 (Fig. 1 in IV) and appeared to be conformational

because mapping attempts using overlapping 16-mer YadA peptides had failed (El

Tahir et al., 2000).

The constructed mutants were tested for their ability to bind serum FH (ELISA and

immunoblotting using goat antiserum against human FH for detection), and their

serum sensitivities were determined in the serum bactericidal assay. One of the

constructed YadA mutants that did not express YadA due to an out-of-frame deletion

within the stalk-coding region, served as a control in all the experiments.


- 65 -

In general, serum-resistant YadA deletion mutants bound FH (Fig. 3 in IV). However,

none of the constructed mutants, even those expressing YadA with broken periodicity,

was completely devoid of FH binding activity. This suggests that there exists no single

site on the YadA stalk that binds FH. The decrease in FH binding was associated with

a decrease in serum resistance for only some mutants (�261-277 and �301-330). One

of these deletions affected the conformation of YadA (�261-277), and this may

underlie the decrease in both FH binding and serum resistance. The other deletion

(�301-330), however, was in a pentadecad register, and in addition, was partially

covered in those mutants whose ability to bind FH remained unaffected. This

observation suggests that a change of distance between the putative FH-binding

subsites on YadA, rather than deletion of the actual YadA site binding FH, results in a

reduction in FH binding.

Most serum-sensitive mutants did not lose their FH-binding ability (�293-322, �308-

323, �323-363, �371-377). Moreover, the FH-binding mode of the mutants was

similar to that of the wild type YadA and involved SCRs of the entire polypeptide

�193-218

� 214-228

�222-236

�231-246

�244-258

�247-262

�252-266

�256-270

�261-277

�259-273

�263-277

�267-281

�271-285

�278-292

�289-318

�293-307

�293-322

�297-326

�301-330

�308-322

�308-323

�319-333

�323-363

�334-348

�349-363

�364-370

�371-377

�378-384

Figure 16. Schematic representation of deletions introduced to the stalk and neck regions ofYadA of Y. enterocolitica O:3 (IV).


- 66 -

chain of FH (Fig. 6 in IV). Although FH bound to YadA mutant proteins displayed

cofactor activity for FI-mediated cleavage of C3b (Fig. 5 in IV), one cannot ignore the

possibility that its ability to interfere with the formation of the alternative pathway

convertases or to accelerate their decay or both remained unaffected. In addition, the

excessive binding of FH by the mutants may result in FH depletion from serum and

thus trigger uncontrolled activation of the complement.

To conclude, FH does not target one particular segment of the YadA stalk. Instead, FH

binding to YadA appears to depend on the recognition of several complementing and

discontinuous structural YadA motifs. Moreover, the spacing of these motifs seems

crucial for the FH binding. The conformational and discontinuous nature of FH

binding has been reported for other proteins predicted to form coiled-coils,

streptococcal M protein, OspE and BBA68 of B. burgdorferi, and FhbA of B. hermsii

(Fischetti et al., 1995; Hovis et al., 2006; McDowell et al., 2004, 2005; Metts et al.,

2003). The structural motifs of these proteins were crucial for forming the binding site

for FH. Distortion of one of the three widely spaced coiled-coils of OspE resulted in

attenuated or completely abolished FH binding (McDowell et al., 2004; Metts et al.,

2003), whereas destabilization of the BBA68 coiled-coils resulted in reduced FH-

binding capacity (McDowell et al., 2005). Since none of the YadA deletions

completely abolished FH-binding ability, one could suggest that when one of the

YadA sites involved in the interaction is deleted, structural motifs elsewhere on the

right-handed part of the YadA stalk, having clear internal symmetry, may find

complementary regions on FH.

4.3.1.2 YadA and C4bp

Several proteins belonging to the family of trimeric autotransporters, such as H.

ducreyi DsrA, and M. catarrhalis UspA1 and UspA2, were shown to bind C4bp

(Abdullah et al., 2005; Nordström et al., 2004). Our receptor-mapping analysis pointed

to YadA as one of the C4bp receptors on Y. enterocolitica. We thus we wanted to

characterize YadA-C4bp interaction in more detail (V).

To show that the interaction between YadA and C4bp is direct, we tested the binding

of serum C4bp to TritonX-114-extracted YadA in affinity blotting. That we found


- 67 -

binding of C4bp to YadA trimer suggests a direct interaction between these proteins

(Fig. 6 in V).

To study whether the interaction between YadA and C4bp is ionic strength-dependent,

we examined the effect of salt on the YadA-C4bp interaction (Fig. 7 in V). Bacteria

expressing YadA were incubated with 125I-C4bp in the presence of salt (50-650 mM).

Because salt inhibits YadA-C4bp interaction at a concentration required to inhibit the

binding of C4bp to C4b (Blom et al., 2000), it is suggested that the positively charged

cluster of amino acids between CCP1 and CCP3, involved in C4b binding, would also

interact with YadA (Accardo et al., 1996; Härdig et al., 1997; Ogata et al., 1993).

Knowing that heparin competes with C4b for C4bp binding, by binding to CCP1-3

(Blom et al., 1999; Hessing et al., 1990; Villoutreix et al., 1998), we examined

whether it could inhibit C4bp-binding to YadA in an analogous fashion. Bacteria were

incubated with 125I-C4bp in the presence of heparin (0-1000 μg/ml), and its influence

on the binding was determined. Heparin weakly inhibited C4bp binding to YadA, and

a relatively high heparin concentration was required for 50% inhibition of the C4bp

binding to YadA (Fig. 7 in V). Although C4b/heparin and YadA do not appear to bind

to exactly the same site on C4bp, one cannot exclude the possibility that these binding

sites overlap.

We examined the functional activity of the YadA-bound C4bp by a cofactor assay. It

clearly showed that C4bp bound to YadA retained its regulatory function (Fig. 2 in V).

4.3.1.3 Ail and FH

Knowing that Ail contributed to FH binding when not blocked by LPS, we wanted to

study whether Ail-bound FH maintains its regulatory function. The cofactor assay

showed that Ail-bound FH was fully functional and displayed cofactor activity for FI-

mediated cleavage of C3b (Fig. 2 in III).

Next, we aimed to examine the nature of the interaction between Ail and FH. For that,

we incubated the single Ail-positive bacteria with purified FH in the presence of salt

(50-650 mM) or heparin (0-1000 μg/ml). That both salt and heparin reduced the FH

binding to Ail (Fig. 5 in III) suggests that the interaction between the proteins is ionic


- 68 -

in nature and involves FH region(s) targeted by heparin, i. e. SCR 7, SCR 13, and SCR

20 (Blackmore et al., 1996, 1998b; Pangburn et al., 1991).

To map in more detail the FH regions involved in interaction with Ail, Ail-expressing

bacteria were incubated with the recombinant FH fragments that cover the whole FH

molecule. The results suggest that the SCR6 and SCR7 may constitute the binding site

for Ail (Fig. 17 and Fig. 4A in III).

4.3.1.3.1 Mapping Ail regions involved in FH binding

To identify the Ail region involved in binding of FH, we substituted amino acids

shown to be crucial for serotype O:8 Ail-mediated serum resistance (Miller et al.,

2001). We thus substituted single amino acids in loop 2 or loop 3, His65Ala and

Ser100Asp, respectively (IV, Fig. 18). Two mutants having double amino acid

substitutions in loop 2, Asp67Gly Leu68Gly and Asp67Ala Leu68Arg, expressed

minimal amounts of Ail and were not studied further (IV, Fig. 18).

Two single substitution mutants were tested for their ability to resist the complement-

mediated killing in the bactericidal assay. As the mutant bacteria showed decreased

resistance both to NHS and to EGTA-Mg serum, we wanted to examine whether their

Figure 18. Schematic representation of the amino acid substitutions introduced to Ailof Y. enterocolitica serotype O:3, loops 1-4 (L1-4).

Figure 17. Schematic representation of FH domains interacting with Ail of Y. enterocolitica

serotype O:3


- 69 -

ability to bind serum FH was affected by the mutations introduced. The mutants

displayed similar levels of bound FH as the wild type (Fig. 5 in IV). This suggested

that the Ail-mediated mechanism of serum resistance does not depend primarily on FH

binding. The biological significance of FH binding by Ail, a factor promoting Yersinia

entry into tissue culture cells in vitro (Miller and Falkow, 1988), could thus be related

to the attachment of bacteria to the glycosaminoglycans and sialic acids on host cells

via FH bridging.

4.3.1.4 Ail and C4bp

Knowing that Ail binds C4bp in the absence of LPS, we wanted to study whether that

the interaction between Ail and C4bp is direct. Binding of serum C4bp to �-

octylglucoside- and TritonX-114-extracted Ail was tested in a ligand blotting assay.

No C4bp binding to Ail could be detected, however, suggesting that its conformational

structure is required for the binding to take place (V).

To characterize the Ail-C4bp binding in more detail we tested whether the binding is

dependent on charge (Fig. 7 in V). Bacteria expresssing Ail were incubated with 125I-

C4bp in the presence of heparin (0-1000 μg/ml) or salt (50-650 mM). Heparin

efficiently and dose-dependently inhibited the binding of C4bp to Ail, suggesting that

CCP1-3 domains of the C4bp �-chain are involved. That the Ail-C4bp interaction was

less salt-sensitive than were YadA-C4bp or C4b-C4bp interactions (Blom et al., 2000)

indicates that the interaction is hydrophobic in nature. Thus, Ail binding sites on C4bp,

although apparently not equivalent, may still overlap with those involved in C4b- or

YadA- C4bp interactions.

To examine whether Ail-bound C4bp remained functionally active, the cofactor assay

was performed. The results clearly showed that C4bp bound to Ail-expressing bacteria

retained its regulatory function (Fig. 2 in V).

CONCLUSIONS AND FUTURE PROSPECTS

- 70 -

5. CONCLUSIONS AND FUTURE PROSPECTS

In complement activation, FH and C4bp are the key regulators that prevent the

complement cascade from attacking host tissues. Some bacteria may bind and deposit

these regulators on their own surfaces and thus provide themselves with an efficient

means to avoid complement activation. In consequence, bacteria resist complement-

mediated lysis and opsonin-dependent phagocytosis.

This study has demonstrated that Y. enterocolitica, similar to many other pathogens,

recruits both FH and C4bp to its surface to ensure protection against the AP- and

CP/LP-mediated killing. YadA and Ail, the most crucial serum resistance factors of Y.

enterocolitica, mediate the binding of FH and C4bp. FH - YadA interaction involves

multiple higher structural motifs on the YadA stalk and the SCRs of the entire

polypeptide chain of FH. The Ail binding site on FH has been located to SCRs 6 and 7.

The binding site for FH on Ail, however, remains undetermined. Both YadA- and Ail-

bound regulators display full cofactor activity for FI-mediated cleavage of C3b/C4b.

FH/C4bp-binding characteristics do, however, differ between YadA and Ail. In

addition, Ail captures the regulators only in the absence of blocking O-ag and OC,

whereas YadA binds FH/C4bp independent of the presence of other surface factors.

Independent of mode of binding, however, YadA and Ail provide Y. enterocolitica a

means to avoid complement-mediated lysis, enhancing chances for the bacteria to

survive in the host during various phases of infection.

To investigate whether complement resistance contributes to pathogenesis of

yersiniosis in vivo would be of major importance. As YadA-mediated collagen binding

is essential for full virulence of Y. enterocolitica (Tamm et al., 1993), mice should be

challenged with serum-sensitive but collagen-binding Y. enterocolitica bacteria,

expressing YadA with the head and neck domains intact. In fact, Ackermann and co-

workers found that serum-sensitive Y. enterocolitica bacteria that express YadA

comprising the translocation unit of Oca family members showed attenuation in mice

(Ackermann et al., 2008). It thus appears that YadA-mediated serum resistance

contributes to bacterial virulence in vivo. It would be interesting, however, to examine

whether the correlation between serum sensitivity and virulence in the mouse model

could be observed also with the serum-sensitive YadA mutants generated in this study,

ones that carry the deletions within the stalk. It would also be of interest to show that

CONCLUSIONS AND FUTURE PROSPECTS

- 71 -

the binding of FH/C4bp by Y. enterocolitica takes place in vivo. FH/C4bp-deficient

mice could serve to study how the lack of the regulators affects the pathogenesis of

yersiniosis.

ACKNOWLEDGEMENTS

- 72 -

ACKNOWLEDGEMENTS

This study was carried out at the University of Helsinki, in the Department of Bacteriology and

Immunology, Haartman Institute. I wish to thank the Graduate School of Biotechnology and

Molecular Biology for their financial support and for organizing interesting courses and

activities. This work has also been supported by the Academy of Finland, Microbes and Man

Program.

Sometimes we forget how we got where we are and the paths we took to get there. I will be

always grateful to my supervisor, Professor Mikael Skurnik, for the opportunity to come to

Helsinki and work in his research group. Thank you for taking a chance on me and for guiding

me into the world of microbiology. I am very grateful for the many insightful discussions we

had and suggestions you gave me. With all your knowledge, you are my primary resource for

getting scientific problems solved.

I would like to thank Professor Seppo Meri for his contribution to the original papers, his great

enthusiasm, unwaveringly positive attitude, and for sharing his wealth of complement

knowledge with me so freely.

I express deep gratitude to my thesis committee follow-up members, Docents Benita

Westerlund-Wikström and Sakari Jokiranta, for their valuable comments concerning my

research project.

Docent Benita Westerlund-Wikström and Professor Ilkka Julkunen earn thanks for

thoughtfully reviewing this thesis on such a tight schedule and providing constructive

criticism.

I am grateful to Carol Norris for thorough linguistic editing of the thesis.

I want to extend my thanks also to other co-authors (alphabetically): Peter Ahrens, Anna

Blom, Tea Blom, José Antonio Bengoechea, Markus Gruber, Jeffrey Hoorfar, Heidi

Hyytiäinen, Hanna Jarva, Vesa Kirjavainen, Andrei Lupas, Peter Lübeck, Camino Pérez-

Gutiérrez, Saara Salmenlinna, and Reija Venho for their invaluable contributions to the

original papers.

I warmly thank the past and present members of the Yersinia lab. I will be always thankful to

José Antonio Bengoechea for his help and advice during my time as an undergraduate student

in Turku. Joséan, you remain my best model for a scientist and teacher. I also thank other

members of the Yersinia lab from the Turku period; Elise Pinta, Claire Pacot-Hiriart, Saija

ACKNOWLEDGEMENTS

- 73 -

Kiljunen, Maria Pajunen, and Yasmin El-Hag El-Tahir, for generating a nice working

atmosphere.

For listening to me babble on and on, and for offering along the way encouragement,

sympathy, good humor, and other forms of sustenance, I thank past and present members of

the Helsinki team; Alexandra Götz, Saara Salmenlinna, Heidi Hyytiäinen, Taina Niskanen,

Nina Akrenius, Pirjo Matero, Tea Blom, Susanna Nykänen, and Chun-Mei Li. I cherish your

friendship and will never forget our many wonderful lunches, “obligatory” coffee/tea breaks,

and fun activities we have done together, including pesäppalo, möllky, and mushroom picking!

I still get a laugh when I reminisce about the time we played “volleyball” on Haukilahti beach

this past summer!

For being a fun bunch with lots of enthusiasm and optimism I thank undergraduate students

visiting the Yerisnia lab, Laura Kalin, Tiina Kasanen, Heli Mönttinen, Katarzyna Frej, Agata

Kosarewicz, Ewelina Przydacz, Monika Rybak, and Magdalena Szeliga. Visiting post-doctoral

fellows Nadezda Fursova, Katarzyna Duda, and Haadi Peeri Dogaheh are thanked for adding a

flavor to Yersinia-lab life.

I thank Anita Saarinen for technical assistance, providing lab essentials, and teaching me how

to count in Finnish (up to 10 though…). Anita introduced suppilovahvero into my cuisine but

despite numerous approaches she failed to develop in me a taste for salmiaki… I still cannot

eat it without choking…

I thank the complement research group for not running away upon seeing me coming into the

lab asking for another “small” favor. I trully appreciate your patience and eagerness to help out

every time I needed it. Special thanks go to those I bothered the most; I thank Hanna Jarva for

her great enthusiasm, valuable comments, and assistance during my very first experiments

performed in the complement lab, Nathalie Friberg for our invigorating chats, and Sami

Junnikala for lending an ear (or two) to listen, and eagerness to help (hey, what about that V8

Mustang ride?).

It has been a pleasure and privilege to work with all the staff of the Department of

Bacteriology and Immunology. I thank Pentti Kuusela for so many laughs, everyday chats,

organizing glögi every year, and that rhubarb pie that sweetened one particularly hectic day at

the lab; Jarmo Juuti for nice chats in the office; Eila Ketolainen for always having a smile for

me, a glögi recipe (a really good one), and help with many practical matters in the lab; Riina

and Malcolm Richardson for their positive and supportive (well… except for the dissertation-

disaster stories) attitude, and Daniela Schönberg-Norio for good humor and for introducing a

concept of laughter yoga to me! I believe we practiced it several times during our coffee/lunch

breaks without truly realizing it! For providing great company for daily commuting to/from

work, and recommending books worth reading, I thank Marjatta Turunen. For admirable

ACKNOWLEDGEMENTS

- 74 -

immense optimism, kindness, openness, and help with all the administrative (and not only)

hitches, I thank Carina Wasström and Kirsi Udueze.

I thank Outi Monni for her friendliness, support, and motivating me to go in for sports (that

was very therapeutic by the way!) when I felt like lazying on my sofa.

I would like to thank my very special friends, Ula Guzik for reminding me not to worry too

much and being close by even when kilometers apart, and Ann Marie Klein for much-needed

reassurance and support in the roughest moments of my life.

My unending gratitude goes to my beloved parents for their boundless love, and support, and

their providing me with a wonderful sense of home and belonging. I thank my parents-in-law

for support and accepting me into their family. I owe thanks to my sister, Agnieszka, for being

my best friend, having a very special knack for cheering me up, for all her advice and support.

I thank my brother-in-law, Rafal, for his friendship, and the little onces, Mateuszek and

Martusia, for the joy, fun, and happiness they have brought to our lives.

I married the best man out there for me. The years of this PhD have not been an easy ride, both

academically and personally. I truly thank you, Grzes, for sticking by my side during my good

and bad times. Only your great love and support helped me survive the phases of mourning the

death of my Mum, when I was irritable and depressed. We both learned a lot about life, and

feel that we strengthened our commitment to each other. I love you very much! Let’s live life

to the fullest!

As I close this section, I am very aware that just as the pyramidion—the single capstone of a

pyramid—is supported by numerous stones, this thesis would not be the same without the help,

support, and contributions of all these above-mentioned people.

Thank you all,

Marta

BIBLIOGRAPHY

- 75 -

BIBLIOGRAPHY

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