Outer Membrane Protein YadA Research Articles

New pathogen-specific immunoPET/MR tracer for molecular imaging of a systemic bacterial infection

The specific and rapid detection of Enterobacteriaceae, the most frequent cause of gram-negative bacterial infections in humans, remains a major challenge. We developed a non-invasive method to rapidly detect systemic Yersinia enterocolitica infections using immunoPET (antibody-targeted positron emission tomography) with [64Cu]NODAGA-labeled Yersinia-specific polyclonal antibodies targeting the outer membrane protein YadA. In contrast to the tracer [18F]FDG, [64Cu]NODAGA-YadA uptake co-localized in a dose dependent manner with bacterial lesions of Yersinia-infected mice, as detected by magnetic resonance (MR) imaging. This was accompanied by elevated uptake of [64Cu]NODAGA-YadA in infected tissues, in ex vivo biodistribution studies, whereas reduced uptake was observed following blocking with unlabeled anti-YadA antibody. We show, for the first time, a bacteria-specific, antibody-based, in vivo imaging method for the diagnosis of a Gram-negative enterobacterial infection as a proof of concept, which may provide new insights into pathogen-host interactions.

Characterization of Complement Factor H Binding toYersinia enterocoliticaSerotype 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.

Molecular mimickry between HLA B27 and Yersinia, Salmonella, Shigella and Klebsiella within the same region of HLA α1-helix

Two new examples of amino acid homology between HLA B27 and microbes triggering HLA B27-associated diseases are described. An outer membrane protein YadA (Yersinia adhesin, previously called Yop1) of Yersinia enterocolitica and Y. pseudotuberculosis shares a linear tetrapeptide with HLA B27. A cationic outer membrane protein OmpH of Salmonella typhimurium shares homology with five amino acids of HLA B27 in a non-linear fashion. The four amino acids of YadA are also notably included in the hexapeptide identical between Klebsiella pneumoniae nitrogenase and HLA B27, and three of them occur in the pentapeptide shared by a Shigella flexneri protein and HLA B27. Antibodies against synthetic peptides including HLA B27 homologues sequences of YadA and OmpH were observed in one-third of the patients with HLA B27 associated diseases. Antibodies were directed against a flanking sequence next to the amino acid sequences shared by arthritis-triggering microbes and HLA B27. The area of identity in each example of this molecular mimicry (Yersinia, Salmonella, Shigella and Klebsiella) is located in the same place on the HLA B27 molecule: between amino acids 70 to 78 in the variable region of alpha 1-helix. This area of HLA B27 molecule includes sites predicted to be important for binding processed antigens.

Anti-recombinant V antigen serum promotes uptake of Yersinia enterocolitica serotype 08 by macrophages.

Phagocytosis resistance even in the presence of opsonizing antibodies is a key feature of pathogenic Yersinia spp. Nevertheless, antibodies against the secreted V antigen and the outer membrane protein YadA are known to mediate protection against Y. enterocolitica serotype 08 in a mouse model with intravenous infection. To investigate the impact of anti-V antigen serum on the interaction of Y. enterocolitica and phagocytic cells, gentamicin kill assays and immunofluorescence staining were performed. In contrast to anti-YadA, the presence of V antigen-specific antibodies resulted in an increased uptake of yersiniae by macrophages. The inhibition of phagocytosis by cytochalasin D suppressed the anti-V antigen-mediated uptake. The uptake-promoting effect of anti-V antigen was more distinct for macrophages than for polymorphonuclear leukocytes. The findings of the passive immunization experiments using an orogastric infection model were in agreement with those of cell-culture experiments. In the first 3 days of infection both antisera exhibit no protective effect on the multiplication of the bacteria in the Peyer's patches. Only mice passively immunized with anti-V antigen survived lethal oral infections with Y. enterocolitica serotype 08. Taken together, the results support the assumption that V antigen might be part of the translocation apparatus and that anti-V antigen inhibits the Yop translocation. In addition, antisera against in-frame-deleted recombinant V antigen were generated. Protection experiments using these antisera suggested that the type-specific region (amino acids 225-232) of the V antigen might not be a protective epitope.

Penetration of M cells and destruction of Peyer's patches by Yersinia enterocolitica: an ultrastructural and histological study.

Yersinia enterocolitica is enteropathogenic for man and rodents. Previous studies provided evidence that Y. enterocolitica invades the lymphoid follicles of the Peyer's patches (PP) of the small intestine. In this study Y. enterocolitica-induced tissue alterations of the follicle-associated epithelium (FAE) and the underlying PP tissue were analysed by scanning (SEM) and transmission electron microscopy (TEM) as well as by conventional histological examination. For this purpose, an experimental mouse infection model including orogastric infections as well as ileal loop experiments were used. A rapid and selective colonisation of the FAE after orogastric yersinia infection was observed by SEM. TEM studies confirmed that Y. enterocolitica adhered closely to the FAE including M cells and enterocytes. Histological studies and TEM revealed that Y. enterocolitica selectively invaded the PP via M cells but not via other cells of the FAE. One day after Y. enterocolitica infection the FAE was altered and small micro-abscesses comprising yersiniae expressing the major outer-membrane protein YadA were observed immediately beneath the FAE. Adjacent villi were dilated from lymphangiectasis and transmigrating polymorphonuclear leucocytes (PMNL) were found within the epithelium. At 5-7 days after infection the FAE and parts of PP were destroyed. Profound alterations of the cyto-architecture of the PP were due to the enormous recruitment of PMNL. By day 5 after infection, abscesses were found in the mesenteric lymph nodes. However, TEM studies revealed evidence that Y. enterocolitica may disseminate from the PP not only via the lymphatics but also by invasion of blood vessels. Taken together, the results of this study demonstrate that the FAE is the primary site of host-pathogen interaction in Y. enterocolitica infection and that this pathogen penetrates M cells and subsequently induces destruction of the PP.

Role of YadA-mediated collagen binding in arthritogenicity of Yersinia enterocolitica serotype O:8: experimental studies with rats

Outer membrane protein YadA, Yersinia adhesin, is one of the plasmid-encoded virulence factors of yersiniae. YadA protects bacteria against host defense through several different mechanisms. One important role of YadA is to mediate binding to several collagen types. Our recent study revealed that a yadA null mutant of Yersinia enterocolitica serotype O:8 has a drastically reduced arthritogenic capacity when injected intravenously into Lewis rats. To further characterize the arthritogenic role of YadA, we repeated the rat experiments with strain Y. enterocolitica O:8/pYV082; this strain expresses a YadA deletion derivative lacking 22 amino acids from the amino-terminal hydrophobic region and does not bind to collagen. Y. enterocolitica O:8/pYV082 induced arthritis in 5 to 14% of rats inoculated with arthritogenic doses, whereas the arthritis incidence with the wild-type parent strain was 65%. The parent strain was slightly more virulent than Y. enterocolitica O:8/pYV082, as determined by rat mortality. It also frequently induced skin abscesses, whereas Y. enterocolitica O:8/pYV082 did not. Infection kinetics in spleen and mesenteric lymph nodes were about the same with both of the bacterial strains used, and the same was true of the Yersinia-specific antibody response. Altogether, these results suggest that YadA-mediated collagen binding contributes to the arthritogenicity of Y. enterocolitica O:8.

Role of Yops in inhibition of phagocytosis and killing of opsonized Yersinia enterocolitica by human granulocytes

The virulence plasmid of Yersinia enterocolitica codes for the production of the outer membrane protein YadA and the secretion of several proteins, called Yops, which may play a role in the interaction between granulocytes and this bacterium. We investigated whether the expression of YadA or the secretion of Yops affected the phagocytosis and killing of opsonized Y. enterocolitica by human granulocytes. The rates of phagocytosis and killing of Y. enterocolitica by granulocytes in suspension in the presence of rabbit Yersinia antibodies and complement were determined by microbiological assays. In addition, noningested cell-adherent bacteria were differentiated from ingested yersiniae by immunofluorescence microscopy. Plasmid-bearing opsonized Y. enterocolitica was able to inhibit phagocytosis and killing by human granulocytes. The inhibition of phagocytosis was specific for the plasmid-bearing strain of Y. enterocolitica, since granulocytes were still able to phagocytose and kill Staphylococcus aureus in the presence of Y. enterocolitica. Plasmid-cured Y. enterocolitica was readily phagocytosed and killed by these cells. To investigate the role of YadA or Yops in the inhibition of phagocytosis by granulocytes, the phagocytosis of mutant strains unable to express YadA or to secrete Yops was studied. A Y. enterocolitica mutant unable to secrete Yops lost its ability to inhibit phagocytosis; a mutant expressing only YadA was readily ingested by granulocytes. These results indicate that after attachment of opsonized Y. enterocolitica to granulocytes, Yops play an important role in inhibiting the ingestion of Y. enterocolitica by human granulocytes.

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.

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 form 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 degrees C. The gel-purified 116-kDa YadA species was completely converted to the 200-kDa species by heating at 100 degrees 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.

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.

Outer membrane protein YadA of enteropathogenic yersiniae mediates specific binding to cellular but not plasma fibronectin.

The binding of bacteria or bacterial products to host proteins of tissue extracellular matrix may be a mechanism of tissue adherence. We investigated interactions of the plasmid-encoded outer membrane protein YadA, which confers pathogenic functions on enteropathogenic yersiniae, with fibronectin. Attachment of YadA-positive and YadA-negative recombinant Yersinia enterocolitica strains to cartilage-derived human cellular fibronectin and human plasma fibronectin in the solid phase revealed that YadA mediates binding of yersiniae to cellular fibronectin in a saturable, concentration-dependent manner. The interaction could be inhibited by an anti-YadA-specific anti-serum. An anti-beta 1-integrin antibody and the synthetic peptide G-R-G-D-S-P, representing the binding site for alpha 5 beta 1-integrin on fibronectin, did not block attachment of YadA-positive yersiniae to cellular fibronectin, indicating a binding site for YadA on cellular fibronectin independent of the R-G-D-S-containing site. By contrast, YadA failed to mediate binding to plasma fibronectin immobilized on nitrocellulose or plastic surfaces. These observations provide evidence for the hypothesis that the binding region for YadA in cellular fibronectin is not present in plasma fibronectin. This study is the first report on differential binding of bacteria to splicing variants of fibronectin. Further experiments might answer the question whether binding of YadA to cellular fibronectin contributes to the pathogenesis of yersiniae, both to the initial adhesion of the bacteria to the matrices of the host and to the arthritogenic potential of enteropathogenic yersiniae.

Role of the Transcription Activator VirF and the Histone-like Protein YmoA in the Thermoregulation of Virulence Functions in Yersiniae

The chromosome of Y. enterocolitica encodes a heat-stable enterotoxin, Yst, being related to STI. The capacity to produce Yst generally disappears during storage of the strains. In these strains, the yst gene is intact but remains silent. The pYV plasmid encodes the eleven secreted antihost proteins called Yops as well as the outer membrane protein YadA. The Yops are secreted by a novel, pYV-encoded secretion mechanism. This mechanism which does not involve the removal of an N-terminal signal sequence, is encoded by the pYV virA and virC loci. The virC locus contains 13 genes called yscA-M. The virA locus encodes the LcrD membrane protein. The yop, yadA and ysc genes form the yop regulon controlled by transcriptional activator VirF. Transcription of the yop, yadA, ysc and virF genes is controlled by temperature. A chromosome-encoded histone-like protein, called YmoA, is involved in the thermoregulation of the yop regulon, which suggests that this thermoregulation could result from temperature-induced changes in DNA topology. The phenotype of ymoA mutants resembles that of osmZ or drdX mutants of E. coli but YmoA is not the Yersinia homologue of the E. coli histone H1. The YmoA histone is also involved in the silencing of the yst gene.

Plasmid-encoded outer membrane protein YadA mediates specific binding of enteropathogenic yersiniae to various types of collagen.

The plasmid-encoded outer membrane protein YadA of enteropathogenic yersiniae is associated with pathogenicity. Recently, collagen binding of YadA-positive yersiniae was reported without detailed characterization (L. Emödy, J. Heesemann, H. Wolf-Watz, M. Skurnik, G. Kapperud, P. O'Toole, and T. Wadström, J. Bacteriol. 171:6674-6679, 1989). To elucidate the nature of collagen binding to YadA, we used a recombinant Yersinia strain expressing the cloned YadA gene. Direct binding of YadA-positive yersiniae to collagens was demonstrated in affinity blot experiments on nitrocellulose filters. A spectrum of collagen types in a wide concentration range were tested for their ability to block binding of 125I-labeled collagen type II to YadA-positive yersiniae. The results indicate a specific binding site(s) for YadA in collagen types I, II, III, IV, V, and XI. In contrast, collagen type VI did not bind to YadA. To characterize the binding site(s) more precisely, isolated collagen chains and cyanogen bromide fragments were investigated. These studies revealed that binding of YadA to collagen type I is confined to the alpha 1(I) chain, whereas the binding site within collagen type XI is localized in the alpha 3(XI) chain. alpha 2(I), alpha 1(XI), and alpha 2(XI) did not bind to YadA. Most interestingly, in the alpha 1(II) chain the specific binding site for YadA resides in the cyanogen bromide fragment CB10. The latter might indicate a binding site that does not depend on conformation. Based on these findings, further fragmentation and the synthesis of peptides may allow definition of the peptide sequence(s) relevant for YadA binding.

Role of the Yersinia outer membrane protein YadA in adhesion to rabbit intestinal tissue and rabbit intestinal brush border membrane vesicles

The Yersinia virulence plasmid confers on strains of Yersinia pseudotuberculosis and Y. enterocolitica an adhesive potential superior to the one encoded by the chromosome alone. We have evaluated the role of the plasmid-encoded outer membrane protein YadA (formerly called Yopl) in adhesion. Insertional inactivation of the yadA gene (formerly called yopA), which encodes YadA, led to a reduction in the capacity of plasmid-carrying strains of Y. pseudotuberculosis 0:III and Y. enterocolitica 0:9 to adhere to intestinal tissue, brush border membranes and polystyrene surfaces. The adhesive characteristics of the mutants were comparable to those of their plasmid-cured counterparts. When the yadA gene from Y. pseudotuberculosis serotype 0:III or Y. enterocolitica serotype 0:3 or 0:8 was cloned into an Escherichia coli strain, increased ability to adhere to intestinal tissue, brush border membrane vesicles and polystyrene was transferred concomitantly. The introduction of the yadA gene from Y. pestis, which is unable to express YadA due to a one base pair deletion, did not change the adhesive characteristics of E. coli. Expression of YadA in the outer membrane may, therefore, make an important contribution to intestinal adherence of the two enteropathogenic members of the Yersinia species, Y. pseudotuberculosis and Y. enterocolitica.

Interactions between Yersinia enterocolitica and rabbit ileal mucus: growth, adhesion, penetration, and subsequent changes in surface hydrophobicity and ability to adhere to ileal brush border membrane vesicles

Interactions between Yersinia enterocolitica and rabbit ileal mucus were examined. Strains carrying the Yersinia virulence plasmid, pYV, adhered to crude mucus but not to intestinal luminal contents that had been immobilized on polystyrene. Using an Y. enterocolitica O:9 mutant in which the yadA gene (formerly called yopA), encoding the high-molecular-weight outer membrane protein YadA (formerly called protein P1 or Yop1), had been inactivated and an Escherichia coli strain carrying the cloned yadA gene, we demonstrated that the ability to adhere to mucus correlated closely to expression of YadA. Thereafter, we evaluated possible consequences of binding between pYV-carrying Y. enterocolitica O:3 strains and constituents in the mucus layer. pYV-carrying strains were able to multiply at a high rate in mucus but not in luminal contents, and the ability to adhere to mucus could therefore facilitate bacterial colonization of the mucosa. However, we also showed in vitro that mucus acted as a barrier for a mucus-adherent, pYV-carrying Y. enterocolitica strain. Furthermore, penetration through, or preincubation with, mucus reduced subsequent adhesion of the pYV-carrying strain to brush border membrane vesicles without simultaneously causing bacterial aggregation. Preincubation with mucus also changed the bacterial surface of the same strain from hydrophobic to hydrophilic. Immunoglobulins present in mucus did not seem to be of importance for our observations. Interaction of Y. enterocolitica with intestinal mucus may thus reflect a host defense mechanism that reduces the pYV-mediated adhesion to the epithelial cell membrane, possibly by rendering the bacteria less hydrophobic.