Role of YadA in arthritogenicity of Yersinia enterocolitica serotype O:8: experimental studies with rats.

Outer membrane protein YadA, the Yersinia adhesin, is one of the plasmid-encoded virulence factors of yersiniae. To evaluate the role of YadA in the pathogenesis of reactive arthritis experimentally, we used YadA- strain YeO8-116, a kanamycin GenBlock insertion mutant derived from Yersinia enterocolitica O:8 wild-type strain 8081. As control strains, a plasmid-cured derivative (8081-c) of 8081 and a YopH- mutant (8081-yoph) were used. In addition, YeO8-116, with the yadA mutation transcomplemented with plasmid pMW10, was used. YeO8-116 induced arthritis to a considerably lesser extent than did wild-type strain 8081 when inoculated intravenously into Lewis rats. In rats surviving for over 14 days after the bacterial inoculation, the arthritis incidences were 6% (4 of 72) among those inoculated with the yadA mutant and 51% (33 of 65) among those inoculated with wild-type strain 8081. When the yadA gene was transcomplemented back to YeO8-116, YeO8-116/pMW10 induced arthritis in 47% (9 of 19) of the inoculated rats. Plasmid-cured strain 8081-c did not induce arthritis in any of the 24 inoculated rats, whereas YopH- mutant 8081-yoph induced arthritis in 20% (5 of 25) of the rats inoculated. Although the 50% lethal dose of YeO8-116 was about sixfold higher than that of 8081, the kinetics of bacterial elimination from the spleen and mesenteric lymph nodes were about the same with both strains. Antibody responses in rats infected with the two strains were also indistinguishable. Our results indicate that YadA contributes to the arthritogenicity of Y. enterocolitica in the rat model.     

Role of the YadA protein in prevention of opsonization of Yersinia enterocolitica by C3b molecules.

When mixed with normal human serum, wild-type pathogenic Yersinia enterocolitica, previously incubated at 37 degrees C, fixed less C3b than its variant cured of the virulence plasmid pYV. Mutants unable to secrete the Yop proteins were still protected against C3b deposition. By contrast, mutants deficient in the production of outer membrane protein YadA fixed more C3b than their YadA+ parent. Gene yadA, cloned as a minimal polymerase chain reaction fragment and introduced in trans, complemented the mutations. Production of YadA by recombinant Escherichia coli LK111 also resulted in a reduction of the amount of C3b deposited on the bacterial surface. The reduction of C3b at the surface of Y. enterocolitica YadA+ compared with YadA- cells correlated with an increase of the amount of factor H fixed at the bacterial surface. The YadA monomer separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane was able to bind factor H. We conclude that factor H bound to YadA reduces the C3b deposition on the bacterial surface, probably by a rapid inactivation of C3b.     

Role of YadA in resistance to killing of Yersinia enterocolitica by antimicrobial polypeptides of human granulocytes.

The virulence plasmid pYVe of Yersinia enterocolitica codes for the production of the outer membrane protein YadA and the secretion of several proteins, called Yops, which may protect this bacterium against killing by human granulocytes. Granulocytes kill ingested microorganisms by oxygen-dependent and oxygen-independent mechanisms, the latter including antimicrobial polypeptides. The aim of this study was to determine whether virulent (pYVe+) Y. enterocolitica and plasmid-cured avirulent (pYVe-) Y. enterocolitica differ in susceptibility to antimicrobial polypeptides extracted from granules of human granulocytes. The acetic acid granule extract contained several polypeptides with antimicrobial activity against Y. enterocolitica as determined by gel overlay and radial diffusion assays. Two of these polypeptides were identified as lysozyme and defensins. pYVe+ Y. enterocolitica was less susceptible than pYVe- Y. enterocolitica to the antimicrobial activity of granule extract, lysozyme, and defensins as determined in a suspension assay, which indicated that the pYVe plasmid mediates a reduced susceptibility to these polypeptides. The role of YadA in the resistance to antimicrobial polypeptides was analyzed by using mutants of Y. enterocolitica that specifically lack or express YadA. The results demonstrated that YadA conferred resistance to the killing of Y. enterocolitica by the granule extract. Together, these results indicate that the plasmid-encoded factor YadA contributes to the resistance of Y. enterocolitica to the killing by antimicrobial polypeptides of human granulocytes.     

Trimer Stability of YadA Is Critical for Virulence of Yersinia enterocolitica

Yersinia adhesin A (YadA) is a trimeric autotransporter adhesin with multiple functions in host-pathogen interactions. The aim of this study was to dissect the virulence functions promoted by YadA in vitro and in vivo. To accomplish this, we generated Yersinia enterocolitica O:8 mutants expressing point mutations in YadA G389, a highly conserved residue in the membrane anchor of YadA, and analyzed their impact on YadA expression and virulence functions. We found that point mutations of YadA G389 led to impaired transport, stability, and surface display of YadA. YadA G389A and G389S mutants showed comparable YadA surface expression, autoagglutination, and adhesion to those of wild-type YadA but displayed reduced trimer stability and complement resistance in vitro and were 10- to 1,000-fold attenuated in experimental Y. enterocolitica infection in mice. The G389T, G389N, and G389H mutants lost trimer stability, exhibited strongly reduced surface display, autoagglutination, adhesion properties, and complement resistance, and were avirulent (>10,000-fold attenuation) in mice. Our data demonstrate that G389 is a critical residue of YadA, required for optimal trimer stability, transport, surface display, and serum resistance. We also show that stable trimeric YadA protein is essential for virulence of Y. enterocolitica.Enteropathogenic Yersinia species Yersinia enterocolitica and Yersinia pseudotuberculosis are food-borne pathogens causing diarrhea, mesenteric lymphadenitis, and reactive arthritis (10). Upon ingestion of contaminated food by the host, the bacteria colonize the intestine and may invade M cells which overlie the Peyer''s patches (PPs) (17). After translocation by M cells, Y. enterocolitica multiplies extracellularly in the adjacent tissue.A major virulence determinant of Y. enterocolitica is the Yersinia adhesin A (YadA) (13). YadA belongs to the family of trimeric autotransporter adhesins (TAAs) (29). TAAs consist of an N-terminal head domain connected via an extended stalk to the C-terminal membrane anchor domain. The head domain of YadA is involved in binding to collagen or host cells, whereas the stalk domain is involved in serum resistance (34). Three membrane anchor domains of one YadA trimer build up a beta-barrel pore, which facilitates the transition of the passenger domains (stalk and head) onto the outer membrane (OM) (21). The exact mechanism of trimeric autotransport remains unclear. There do exist several models, among which the hairpin and threading models propose that the membrane anchor domains integrate into the OM and build up a beta-barrel pore. After that, the passenger domains traverse the pore, starting with either the N or C terminus (5).Numerous efforts have attempted to elucidate the function of YadA in establishing infection with Y. enterocolitica. The domain-function relationships of YadA have been analyzed in detail (35, 36). There is striking evidence that the head domain of YadA is involved in binding to extracellular matrix proteins, such as collagen, and in binding to neutrophils (14, 20, 27, 34). Also, the autoagglutination capacity seems to involve at least parts of the head domain (44). In addition to adherence and autoagglutination, serum resistance seems to be an important function mediated by YadA for virulence of Y. enterocolitica in vivo. Via the binding of serum complement factor H and C4 binding protein (C4BP), YadA may prevent deposition of C3b on the bacterial surface and therefore block formation of membrane attack complexes and killing of the bacteria (6, 23). However, this function has so far been mapped only for factor H, which binds to the stalk domain of YadA (7). The binding region for C4BP is still unknown.Recent studies by Ackermann et al. (1) have shown that only the factual membrane anchor domain of YadA is able to confer serum resistance and mouse virulence to Y. enterocolitica expressing chimeric fusion proteins of the N-terminal YadA passenger domain and the C-terminal membrane anchor domains of the TAAs UspA1 (Moraxella catarrhalis), EibA (Escherichia coli), and Hia (Haemophilus influenzae).We previously showed that YadA and most other known TAAs have a G residue in the second beta-strand of the membrane anchor domain which forms the beta-barrel translocator pore; in the exceptional TAAs, it is either A, S, T, or N (19). This residue (G389 in YadA of Y. enterocolitica O:8) faces the lumen of the beta-barrel. According to a YadA beta-barrel model that was generated based on the Haemophilus influenzae Hia three-dimensional (3D) structure, the free space around G389 should be large enough to accommodate either A, S, C, D, P, N, T, V, E, or Q (in ascending order of side chain volume) without constraining the conformation of adjacent residues. An H residue would reach the limit of the pocket size but might still be accommodated with only minor local structural adjustments. We previously replaced G389 with the four naturally occurring residues (A, S, T, and N) and also with H in order to explore the upper size limit of the pocket. The side chain size of the residues increases in the order G < A < S < T < N < H, and polarity increases in the order A < G < S < T < N < H. When expressed in E. coli, these G389 substitutions were shown to influence YadA surface display and stability, to different degrees (19).To elucidate which of the many functions of YadA in host-pathogen interaction in vitro are of relevance for the pathogenicity of Y. enterocolitica in vivo, we introduced the G389 substitution mutations into the Y. enterocolitica background and thoroughly analyzed trimer stability, accessibility by trypsin, outer membrane localization, adhesion to collagen and HeLa cells, Yop-mediated suppression of cytokine responses, autoagglutination, serum resistance, binding of complement regulatory factors (CRFs), and virulence in mice. From our results, we conclude that the stability of YadA trimers is decisive for the YadA-mediated serum resistance and virulence of Y. enterocolitica in mice.     

Cell-Mediated Immunity to Yersinia enterocolitica Serotype 3 in Patients with Thyroid Diseases

The cellular immunity to Yersinia enterocolitica serotype 3 and crude human thyroid extract in 64 patients with thyroid diseases and 25 controls was studied by the leucocyte migration test. In the patient group as a whole and in patients with Graves' disease and nontoxic diffuse goitre a significantly reduced leucocyte migration towards Yersinia was found when compared with the controls. In controls the migration index was not related to the presence or titre of circulating yersinia antibodies, whereas the migration index of patients with yersinia antibodies was lower than the migration index of patients without yersinia antibodies as well as that of the controls. The leucocyte migration inhibition in two patients with recent yersiniosis was normal during the recovery phase.
In the presence of thyroid extract leucocyte migration inhibition differed only significantly in Graves' disease. However, a significantly positive correlation between inhibition of migration by thyroid extract and by Yersinia was found, while no correlation could be demonstrated in the controls.
The cell-mediated immunity towards Yersinia in thyroid diseases thus demonstrated adds further evidence to the association between Yersinia and thyroid disease.     

Lipopolysaccharide O side chain of Yersinia enterocolitica O:3 is an essential virulence factor in an orally infected murine model.

The rfb gene cluster of Yersinia enterocolitica O:3, responsible for the biosynthesis of the O side chain, was previously cloned, and a Y. enterocolitica O:3 side chain-specific bacteriophage (phi YeO3-12) was isolated (A. Al-Hendy, P. Toivanen, and M. Skurnik, Microb. Pathog. 10:47-59, 1991). This paper describes the isolation and characterization of the bacteriophage phi YeO3-12-resistant mutant of Y. enterocolitica O:3, YeO3-R2. Lipopolysaccharide isolated from YeO3-R2 lacked the O side chain, as evidenced by silver staining and by immunoblots probed with a Y. enterocolitica O:3 O side chain-specific monoclonal antibody. The core was complete, as shown in immunoblots probed with an outer core-specific monoclonal antibody. In Southern blotting with the cloned Y. enterocolitica O:3 rfb region as a probe, there was no detectable difference in the hybridization pattern of chromosomal DNA isolated from YeO3-R2 and that isolated from wild-type Y. enterocolitica O:3. This suggests that a point mutation, rather than a large deletion, was responsible for the rough phenotype of YeO3-R2. The virulence of YeO3-R2 was determined in an orally infected desferal-attenuated murine model. The mutant was approximately 50-fold less virulent than the isogenic wild type. The ability of YeO3-R2 to reexpress O side chain, and hence full virulence, was reconstituted by complementing the chromosomal mutation in trans with the distal 6.5 kb of the Y. enterocolitica O:3 rfb region. This same 6.5-kb fragment transcomplemented a transposon mutation in the same area of the Y. enterocolitica O:3 rfb region when expressed in Escherichia coli. This transcomplementation implies that the rfb region of Y. enterocolitica O:3 is organized into at least two separate operons.     

Serum Resistance of Yersinia enterocolitica Expressed in Absence of Other Virulence Markers

Serum resistance of Yersinia enterocolitica after growth at 37 and 25°C appears to be specific for serogroup O:3 and appears to be expressed even in the absence of other phenotypic virulence-associated markers, such as the presence of V antigen, autoagglutination, and calcium dependency after growth at 37°C.     

Yersinia enterocolitica serotype O:3 is arthritogenic for mice

It is shown, for the first time, that Yersinia enterocolitica serotype O:3 is experimentally arthritogenic. Moreover, it is arthritogenic for the mouse, an optimal model for human yersiniosis. This arthritis can be induced by the oral route, the most common route in man. The pattern of joint disease closely parallels that of human reactive arthritis associated with this pathogen.     

Temperature-modulated immunogenicity to Yersinia pestis from Yersinia enterocolitica O3.

The ability of Yersinia enterocolitica O3, grown at 25 degrees C, to promote cross-immunity to Y. pestis was lost after repeated subcultures at 37 degrees C, which selected for bacterial populations having lower in vivo survival. Subculturing Y. enterocolitica O3 from 37 to 25 degrees C restored the cross-immunogenicity although the in vivo survival remained low.     

Pathogenicity of Yersinia enterocolitica serotype O3 biotype 3 strains

It is known that Yersinia enterocolitica infection in Japan is caused mainly by serotype O3 biotype 4 strains. Recently, however, a number of serotype O3 strains which were classified biotype 3 and which ferment lactose and xylose, instead of sorbose, and give a negative Voges-Proskauer reaction have been isolated from both humans and animals. In this study, comparisons of four properties were made among isolates of Y. enterocolitica serotype O3 biotype 3 from humans, pigs, dogs, cats, and rats and the laboratory stock strains of Y. enterocolitica biotype 4. All strains were tested for the presence of plasmids, calcium-dependent growth at 37 degrees C, autoagglutination activity at 37 degrees C, and recovery of the organisms from the stools of intravenously challenged mice. Biotypes 3 and 4 were positive for these four properties. Plasmid digestion with restriction endonucleases showed the same digestion patterns in both biotypes. These results suggest that Y. enterocolitica serotype O3 biotype 3 strains are pathogenic, as are biotype 4 strains.     

Characterization of different oligomeric species of the Yersinia enterocolitica outer membrane protein YadA

The oligomeric structure of the plasmid-encoded outer membrane protein YadA of Yersinia enterocolitica was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sucrose gradient sedimentation, respectively. The apparent molecular weight (M _r) of the oligomeric 200-kDa YadA species detected by SDS-PAGE varied from 152,000 to 240,000 depending on the respective acrylamide concentration. The atypical electrophoretic behavior of the 200-kDa YadA species results from an exceptionally high relative free mobility as revealed by the Ferguson plot. In contrast, the apparent M _r of 53,000 of the YadA monomer was independent of the acrylamide concentration. An additional oligomeric 116-kDa YadA species was detected by SDS-PAGE when membrane preparations of Y. enterocolitica were solubilized in SDS at 37 °C. The gel-purified 116-k Da YadA species was completely converted to the 200-kDa species by heating at 100 °C and to the monomeric form (M _r 53,000) by heating in the presence of 10 M urea without reducing agents, respectively. This suggests that the 116-kDa YadA species represents the native oligomeric form of YadA, whereas the 200-kDa species is only generated from native YadA during denaturation in SDS. The significance of the 116-kDa YadA species is also supported by the rather slow sedimentation at about 6 S of detergent-solubilized YadA in sucrose gradients, which probably contains only two or three monomers.     

YadA mediates specific binding of enteropathogenic Yersinia enterocolitica to human intestinal submucosa.

The binding of live Yersinia enterocolitica to frozen sections of human intestine was investigated qualitatively by monitoring the binding of bacteria by using Gram or immunoperoxidase staining as well as quantitatively by a new enzyme immunoassay-on-slide method. We have demonstrated that the binding of various Y. enterocolitica serotypes and Escherichia coli clones to frozen sections of human intestine is mediated by the Yersinia adhesin, YadA. The YadA-mediated binding occurs mainly at the submucosal layer of the intestinal wall and only to a limited extent at the mucosal layer; there binding is mostly to the mucin threads. In addition, partially purified YadA binds to frozen sections with a pattern similar to that of intact bacteria. Collagen, laminin, or partially purified YadA only partially inhibited the YadA-mediated binding of bacteria, presumably because YadA is multifunctional. A combination of collagen and laminin inhibited the binding more efficiently. Therefore, YadA may be involved in the interactions with the extracellular matrix molecules after the invasion of the intestinal tissue.     

Serum opsonic capacity against Yersinia enterocolitica O:3 in yersiniosis patients with or without reactive arthritis.

The opsonic capacity of 45 sera from patients with reactive arthritis after Yersinia enterocolitica O:3 infection and of 45 matched sera from yersiniosis patients without post-infection complications was studied at 1-3 months, 5-8 months and 12-20 months after the onset of the infection. Antibody-mediated opsonization of virulence-plasmid-containing Y. enterocolitica O:3 was studied by measuring complement-fixation on opsonized bacteria and opsonophagocytic function of the polymorphonuclear leucocytes (PMN). The PMN response against bacteria pre-opsonized by heat-inactivated sera was measured by using a chemiluminescence (CL) assay. The fixation of complement Clq and C3 on bacteria was determined by flow cytometry using fluorescein-conjugated Clq- and C3c-antisera. All the sera were strongly opsonic at the onset of the infection, and this capacity persisted in most of the patients still at the end of the follow-up. No difference was observed in complement-fixing capacity between the sera of the two groups, but the sera from arthritic patients showed stronger augmentation of PMN CL response at the early phase of the infection (P = 0.005 in the presence of complement, P = 0.04 in the absence of complement). These results suggest that enhanced opsonic capacity may play a role in the development of Yersinia-triggered reactive arthritis by leading to strong activation of the PMN and, consequently, to release of inflammatory mediators.     

Monocytes That Have Ingested Yersinia enterocolitica Serotype O:3 Acquire Enhanced Capacity To Bind to Nonstimulated Vascular Endothelial Cells via P-Selectin

Reactive arthritis is usually a self-limiting polyarthritis which develops after certain gastrointestinal or urogenital infections. Microbial antigens found in the inflamed joints are thought to play a key role in the development of this disease. It is not known how antigens of the pathogenic organisms migrate from the mucosal tissues into the joints. The data presented here show that mononuclear phagocytes which mediate the dissemination of several intracellular pathogens acquire an enhanced capacity to bind to nonstimulated vascular endothelial cells after phagocytosis of Yersinia enterocolitica O:3, one of the causative organisms of reactive arthritis. The increased binding to previously nonstimulated endothelial cells was mediated by P-selectin, whose translocation to the endothelial cell surface was induced by monocytes with intracellular Yersinia bacteria. These results suggest that mononuclear phagocytes may be responsible for the dissemination of bacterial antigens and the initiation of the joint inflammation in reactive arthritis.     

Antigenic identity of Yersinia enterocolitica serotypes O:Tacoma and O21

Yersinia enterocolitica serotypes O:Tacoma and O:21 are antigenically identical and include both pathogenic and nonpathogenic types.     

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.     

Detection of Yersinia enterocolitica serogroup O:3 by a PCR method.

Yersinia enterocolitica is the etiologic agent of a range of clinical situations in humans, but only a small number of serotypes are involved. Among these, Y. enterocolitica O:3 is the most frequently implicated. A PCR method was developed to detect Y. enterocolitica O:3. For this purpose, two pairs of primers were designed to amplify two fragments of the rfb cluster of Y. enterocolitica O:3: a 253-bp fragment of the rfbB gene and a 405-bp fragment of the rfbC gene. A specific detection was obtained only with rfbC primers, which yielded a PCR product of the expected size exclusively with pathogenic Y. enterocolitica of serotype O:3. This pair of primers was combined with the ail, inv, and virF primers previously described (H. Nakajima, M. Inoue, T. Mori, K.-I. Itoh, E. Arakawa, and H. Watanabe, J. Clin. Microbiol. 30:2484-2486, 1992) to allow both the detection and the differentiation between Y. pseudotuberculosis, pathogenic Y. enterocolitica of serotype O:3 and other pathogenic Y. enterocolitica.     

Isolation and use of eight phages for typing Yersinia enterocolitica O3

Subdivision of 137 isolates of Yersinia enterocolitica O3 into eight phagovars has been achieved. Some geographical differences were found in the sources of these phagovars and also of two biovars.