Molecular Details of Serum Resistance of Yersinia enterocolitica

In complement activation, Factor H (FH) and C4b-binding protein (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 complement-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 short consensus repeats (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 lipopolysaccharide O-antigen and outer core, 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.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. This study has demonstrated that an enteropathogic bacterium Y. enterocolitica binds host complement regulators to avoid complement-mediated lysis, enhancing chances for the bacteria to survive in the host during various phases of infection

Yersinia enterocolitica Serum Resistance Proteins YadA and Ail Bind the Complement Regulator C4b-Binding Protein

Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core (OC) do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp), an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host

Role of YadA, Ail, and Lipopolysaccharide in Serum Resistance of Yersinia enterocolitica Serotype O:3

Complement attack is a host strategy leading to elimination of pathogens. Yersinia enterocolitica expresses several potential complement resistance factors: the outer membrane proteins YadA and Ail as well as lipopolysaccharide (LPS). To study the contribution of these factors to the survival of Y. enterocolitica serotype O:3 in nonimmune human serum, we constructed 23 mutant strains of Y. enterocolitica O:3 expressing different combinations of YadA, Ail, LPS O antigen, and LPS outer core. Survival of bacteria was analyzed in normal serum (with functional classical, lectin, and alternative complement activation pathways) and EGTA-Mg-treated serum (only alternative pathway functional). Kinetic killing tests revealed that the most potent single-serum resistance factor needed for long-term survival was YadA; Ail was also indispensable, but it provided short-term survival and delayed the bacterial killing. On the contrary, the LPS O antigen and outer core, when in combination with YadA, Ail, or both, had a minor and often negative effect on serum resistance. Bacteria in the exponential phase of growth were more resistant to serum killing than stationary-phase bacteria. After exposing bacteria to EGTA-Mg-treated serum, O antigen could prevent deposition of covalently bound C3b on bacteria at 3 min of incubation, even as a single factor. At later time points (15 and 30 min) it had to be accompanied by YadA, Ail, and outer core. In normal serum, the bacteria were less resistant to C3b deposition. However, no direct correlation between the C3 deposition pattern and bacterial resistance was observed

Characterization of Complement Factor H Binding to Yersinia enterocolitica Serotype O:3▿

A number of bacteria bind factor H (FH), the negative regulator of the alternative complement pathway, to avoid complement-mediated killing. Here we show that a gram-negative enteric pathogen, Yersinia enterocolitica serotype O:3, uses two virulence-related outer membrane (OM) proteins to bind FH. With Y. enterocolitica O:3 mutant strains displaying different combinations of surface factors relevant to complement resistance, we demonstrated that the major receptor for FH is the OM protein YadA. Another OM protein, Ail, also contributes to FH binding provided that it is not blocked by distal parts of the lipopolysaccharide (i.e., the O antigen and the outer core hexasaccharide). Importantly, we demonstrated that surface-bound FH was functional; both YadA- and Ail-bound FH displayed cofactor activity for factor I-mediated cleavage of C3b. With truncated recombinant FH constructs, we located the binding site of Ail specifically to short consensus repeats 6 and 7 of FH, while YadA showed a novel type of FH-binding pattern and appears to bind FH throughout the entire FH molecule. We thus conclude that Y. enterocolitica, via YadA and Ail, recruits functionally active FH to its surface. FH binding appears to be an important mechanism of the complement resistance of this pathogen

Functional Mapping of YadA- and Ail-Mediated Binding of Human Factor H to Yersinia enterocolitica Serotype O:3▿

Yersinia enterocolitica is an enteric pathogen that exploits diverse means to survive in the human host. Upon Y. enterocolitica entry into the human host, bacteria sense and respond to variety of signals, one of which is the temperature. Temperature in particular has a profound impact on Y. enterocolitica gene expression, as most of its virulence factors are expressed exclusively at 37°C. These include two outer membrane proteins, YadA and Ail, that function as adhesins and complement resistance (CR) factors. Both YadA and Ail bind the functionally active complement alternative pathway regulator factor H (FH). In this study, we characterized regions on both proteins involved in CR and the interaction with FH. Twenty-eight mutants having short (7 to 41 amino acids) internal deletions within the neck and stalk of YadA and two complement-sensitive site-directed Ail mutants were constructed to map the CR and FH binding regions of YadA and Ail. Functional analysis of the YadA mutants revealed that the stalk of YadA is required for both CR and FH binding and that FH appears to target several conformational and discontinuous sites of the YadA stalk. On the other hand, the complement-sensitive Ail mutants were not affected in FH binding. Our results also suggested that Ail- and YadA-mediated CR does not depend solely on FH binding

Bacteria and plasmids used in this work.

<p>HUSLAB, Helsinki University Central Hospital, Helsinki, Finland.</p

Characterization and Biological Role of the O-Polysaccharide Gene Cluster of Yersinia enterocolitica Serotype O:9▿

Yersinia enterocolitica serotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) in Y. enterocolitica O:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer of N-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of the gnd gene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster, galF and galU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC

Binding of serum C4bp to the wild type and YadA-negative <i>Y. enterocolitica</i> serotypes O:3 and O:8.

<p>Wild type bacteria (Ye03 and 8081) and their YadA-negative derivatives (YeO3-028 and YeO8-116, respectively) (3×10⁸) were incubated in 5% heat-inactivated serum (HIS) for 30 min. Bacteria washed in 1/3 PBS were subjected to elution with PBS while those washed with PBS were subjected to elution with 0.1 M glycine-HCl (pH 2.7). The 1/3 PBS or PBS wash fractions (w1 and w2, respectively) and PBS or 0.1 M glycine-HCl (pH 2.7) elute fractions (e1 and e2, respectively) were separated by 8% non-reducing SDS-PAGE and analyzed by immunoblotting using a sheep anti human-C4bp antiserum.</p

Cofactor activity of <i>Y. enterocolitica</i>-bound C4bp for C4b cleavage.

<p><i>Y. enterocolitica</i> serotype O:3 wild-type bacteria (YeO3), YadA-negative less virulent biotype 1A strain 27675 (BT 1A) and Ail-expressing strain YeO3-028-OCR were preincubated with C4bp and after extensive washings exposed to factor I and C4b. C4b and its cleavage products from the supernatants were detected using polyclonal antibodies against C4c. As positive and negative controls, assays containing C4b and FI with or without C4bp, respectively, were included. Inactivation of C4b is demonstrated by the appearance of C4b cleavage fragments (indicated with arrows).</p