Yersinia enterocolitica: subversion of adaptive immunity and implications for vaccine development

Enteric Yersinia spp. invade Peyer's patches, disseminate to lymphoid tissues, and induce mucosal and systemic immune responses. Many virulence factors of Yersinia enterocolitica have been investigated in detail and were found to act on host cells involved in innate and adaptive immunity. Recent work explored as to whether attenuated Y. enterocolitica or recombinant components of Y. enterocolitica can be used as tools for vaccination. We and others have tested whether by means of the type three secretion system in attenuated Y. enterocolitica strains antigens might be delivered to antigen-presenting cells in order to induce CD8 and CD4 T cell responses. Alternatively, recombinant components of Y. enterocolitica such as invasin protein which binds to β1 integrins of host cells have been tested for their ability to target antigen along with microparticles (fused to invasin) to antigen-presenting cells and to act as adjuvant. The work summarized in this article demonstrates that Y. enterocolitica and its components might be useful tools for novel vaccination strategies; in fact, invasin when fused to antigen and coated to microparticles might induce both CD4 and CD8 T cell responses. Likewise, attenuated Y. enterocolitica live carrier strains were reported to induce both CD8 and some CD4 T cell responses. However, we need to know more about how Y. enterocolitica subverts functions of antigen-presenting cells in order to design mutants with optimized antigen delivery features and deletion in those virulence factor that contribute to subversion of innate or adaptive immune responses.     

Attenuated Yersinia enterocolitica mutant strains exhibit differential virulence in cytokine-deficient mice: implications for the development of novel live carrier vaccines

Yersinia enterocolitica mutant strains, including mutants deficient in the chaperone SycH resulting in a functional deficiency in tyrosine phosphatase (YopH), Mn-cofactored superoxide dismutase (SodA), iron-repressive protein 1 (IRP-1), and Yersinia adhesin A (YadA), were demonstrated to be highly attenuated in wild-type C57BL/6 mice. TNFRp55(-/-), IL-12p40(-/-), and IL-18(-/-) mutant mice, in which the Yersinia wild-type strain causes severe systemic infections, were used to investigate whether these Yersinia mutant strains would be attenuated in immunodeficient hosts. A plasmid-cured Yersinia mutant strain was unable to colonize any of the mutant mice tested. A SycH-deficient mutant strain colonized intestinal tissues of these mice but was attenuated for systemic infection in all of the mutant mice. Both YadA- and Irp-1-deficient Yersinia mutants were still attenuated in IL-12(-/-) and IL-18(-/-) mice but were pathogenic in TNFRp55(-/-) mice. By contrast, a Yersinia sodA mutant was highly pathogenic for TNFRp55(-/-) and IL-12p40(-/-) mice while interleukin-18 (IL-18) was dispensable. This finding demonstrates that certain virulence factors enable yersiniae to compete with distinct cytokine-dependent host defense mechanisms. Moreover, while gamma interferon mRNA expression did not reflect protective host responses in cytokine-deficient mice, IL-10 expression coincided with a heavy splenic bacterial load and was associated with progressive infection courses. We can thus segregate minor (SodA), intermediate (YadA and IRP-1), and major (YopH) virulence factors of Y. enterocolitica. Finally, we demonstrate that, even in immunocompromised hosts, Yersinia sycH and, with some restrictions, irp-1 mutants may be suitable for use as live carrier vaccines.     

Evaluation of enzyme immunoassay for the detection of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis strains.

To determine the virulence plasmid-harboring strains of Yersinia enterocolitica, we prepared antiserum against plasmid-encoded proteins of Y. enterocolitica serotype O3 and carried out an enzyme immunoassay (EIA) against temperature-inducible released proteins. This serum reacted with proteins released from not only a Y. enterocolitica serotype O3 strain but also Y. enterocolitica serotype O5:27, O8, and O9 and Y. pseudotuberculosis serotype 1b, 2a, 2b, 2c, 3, 4a, 4b, 5a, 5b, 6, 7, and 8 strains, which all harbored plasmids. Plasmid-cured Y. enterocolitica and Y. pseudotuberculosis strains did not react in the EIA, nor did nonpathogenic Y. enterocolitica strains or Y. frederiksenii, Y. intermedia, and Y. kristensenii strains. These observations demonstrated that this EIA was useful for determining whether the isolated Yersinia strains were pathogenic or not.     

Effect of low- and high-virulence Yersinia enterocolitica strains on the inflammatory response of human umbilical vein endothelial cells

Pathogenic strains of Yersinia spp. inject a set of Yop effector proteins into eukaryotic cells by using a plasmid-encoded type III secretion system. In this study, we analyzed the inflammatory response of human umbilical vein endothelial cells (HUVECs) after infection with different Yersinia enterocolitica strains. We found that both expression of intercellular adhesion molecule 1 and release of the cytokines interleukin-6 (IL-6) and IL-8 by HUVECs are downregulated in a YopP-dependent way, demonstrating that YopP plays a major role in the inflammatory response of these cells. Infection of HUVECs with several low-virulence (biotype 2, 3, and 4) and high-virulence (biotype 1B) Y. enterocolitica strains showed that biotype 1B isolates are more efficient in inhibiting the inflammatory response than low-virulence Y. enterocolitica strains and that this effect depends on the time of contact. We extended the results of Ruckdeschel et al. and found that on the basis of the presence or absence of arginine-143 of YopP (K. Ruckdeschel, K. Richter, O. Mannel, and J. Heesemann, Infect. Immun. 69:7652-7662, 2001) all the Y. enterocolitica strains used fell into two groups, which correlate with the low- and high-virulence phenotypes. In addition, we found that high-virulence strains inject more YopP into the cytosol of eukaryotic target cells than do low-virulence strains.     

Comparison of 2 methods of hemagglutination tests for antibodies against strains of Yersinia enterocolitica and Yersinia pseudotuberculosis

The authors compare results of the assessment of antibodies against bacterial strains of Yersinia enterocolitica and Yersinia pseudotuberculosis. The examinations were made by the haemagglutination technique. In the reaction of the antigen human and ram erythrocytes were used as carriers. The work comprised 462 examinations of patients with an assumed Yersinia aetiology of the disease and 200 healthy controls. The values obtained during parallel examinations with antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis linked to both types of blood cells were processed by statistical methods. It was revealed that when human and ram erythrocytes are used, the antibody levels correlate but are not identical. In the conclusion the authors recommend to unify the methods of different laboratories to facilitate comparison of results.     

Evaluation of iron dextran and mucin for enhancement of the virulence of Yersinia enterocolitica serotype O:3 in mice.

The pathogenic role of Yersinia enterocolitica serotypes O:3, O:8, and O:9 in human infections is well documented. Whereas the virulence of the O:8 strains can be readily demonstrated in mice by 50% lethal dose determinations, the O:3 and O:9 strains have no lethal effect on mice by any route of inoculation. A mouse virulence test for the O:3 and O:9 strains is described. Y. enterocolitica strains were first tested for the presence of virulence-associated plasmid characteristics by auto-agglutination and gel electrophoresis procedures before mouse virulence determinations. The 50% lethal dose of the O:3 strains injected intraperitoneally with 2.5% mucin was about 10(7) colony-forming units. However, histological examinations showed that mucin allowed the growth of Y enterocolitica on the surface of the livers and spleens of the mice without internal lesions. The 50% lethal dose of the same O:3 strains injected intraperitoneally with 1 ml of 10% iron dextran in saline was about 10(5) to 10(6) colony-forming units, and the nonlethal infective dose with typical lesion development was 20 to 200 colony-forming units. The infected mice developed symptoms and extensive liver and spleen lesions which differed from those in mice infected intraperitoneally with the virulent O:8 strains. These results showed that the virulence of the O:3 Y. enterocolitica strains can be measured by intraperitoneal injection with iron dextran. This procedure was used to test the virulence of food isolates, plasmidless strains, and the effect of growth temperatures.     

Differentiation of Yersinia enterocolitica serotype O:5,27 strains by phenotypic and molecular techniques.

Restriction endonuclease analyses of virulence plasmid DNA (REAP) and chromosomal DNA and other phenotypic characteristics were used to study the differentiation of Yersinia enterocolitica serotype O:5,27 strains. There was a close correlation between REAP patterns and the geographical distribution of serotype O:5,27. Human isolates produced only one REAP pattern, which was also found with isolates from pigs and dogs.     

Analysis of the Yersinia enterocolitica 0:8 V antigen for cross protectivity

The plasmid encoded V antigen (Vag) of pathogenic Yersinia spp. is a major virulence factor as well as a protective immunogen. Recently, two main types of Vag, represented by either Yersinia enterocolitica 0:8 or Yersinia pseudotuberculosis, have been identified and it has been suggested, that antibodies generated against one type are unable to protect against Yersinia spp. carrying the other type. By using a recombinant Vag (rVagHis) of the Y. enterocolitica 0:8 type we show here, that actively immunized mice were completely protected against challenge with both, Y. enterocolitica 0:8 and Y. pseudotuberculosis serotype III. In addition, passive protection was possible with polyclonal rabbit anti-rVagHisIgG. However, while a single antibody dose (200 microgramg) was sufficient to protect against challenge with Y. enterocolitica 0:8, repetitive injections at intervals of 2 to 3 days were needed to protect against challenge with Y. pseudotuberculosis III. The apparent difference in protection correlated with a rapid disappearance of anti-rVagHisIgG from the circulation by days 3 to 4. The data therefore indicate, that expression of distinct types of Vag by Yersinia spp. does not necessarily exclude immunoprotection in mice immunized with the other type of Vag. It rather appears, that differences in immunoprotection between Yersinia species relate to the amount of cross-protective antibody. Finally, as revealed by the lack of complement-mediated killing and the lack of immunostaining of Yersiniae with anti-rVagHisantibodies, evidence is provided to indicate that immunoprotection does not occur via opsonisation or complement lysis.     

Influence of gastrointestinal commensal bacteria on the immune responses that mediate allergy and asthma

The human intestine contains more than 100 trillion microorganisms that maintain a symbiotic relationship with the host. Under normal conditions, these bacteria are not pathogenic and in fact confer health benefits to the host. The microbiota interacts with the innate and adaptive arms of the host's intestinal?mucosal immune system and through these mechanisms drives regulatory cell differentiation in the gut that is?critically involved in maintaining immune tolerance. Specifically, the microbiota can activate distinct tolerogenic dendritic cells in the gut and through this interaction can drive regulatory T-cell differentiation. In addition, the microbiota is important in driving T(H)1 cell differentiation, which corrects the T(H)2 immune skewing that is thought to occur at birth. If appropriate immune tolerance is not established in early life and maintained throughout life, this represents a risk factor for the development of inflammatory, autoimmune, and allergic diseases.?Early-life events are instrumental in establishing the microbiota, the composition of which throughout life is influenced?by various environmental and lifestyle pressures. Significant efforts are now being made to establish interventional approaches that can create a healthy microbiota that confers maximum tolerogenic immunomodulatory effects in the gut and that will protect against systemic inflammatory disease pathologies.     

Comparison of broth microdilution and disk diffusion test for antimicrobial resistance testing in Yersinia enterocolitica 4/O:3 strains

The aim of this study was to compare the results obtained from two methods for the determination of antimicrobial resistance in 110 Yersinia enterocolitica 4/O:3 strains. Ten antimicrobial agents were tested using broth microdilution and disk diffusion. Similar results were determined for six antimicrobials. Very major errors (false-susceptible by disk diffusion test) were detected for ampicillin (at a rate of 1.8%). Major errors (false-resistant by disk diffusion test) were found for streptomycin (0.9%) and sulfamethoxazole (1.8%). Minor errors (intermediate susceptible by disk diffusion and resistant or susceptible by microdilution) were obtained for ampicillin (2.7%) and sulfamethoxazole (13.6%). All Y. enterocolitica were resistant to at least one antimicrobial agent. Resistances to three classes of antimicrobial agents were obtained by 3% of the strains included in the study. A slightly higher frequency of multiresistance was obtained by disk diffusion (3%) compared with broth microdilution (1%). Resistance to streptomycin was found frequently (13%); in contrast, resistance to tetracycline was rare (1%). The disk diffusion test produced unacceptably high rates of very major errors for ampicillin and a high frequency of minor errors for sulfamethoxazole. Susceptibility tests should thus be carried out by the more reliable method of microdilution. Most of the antimicrobials that can be used for therapy were very effective when tested against Y. enterocolitica. In order to identify changes in susceptibilities as early as possible, antimicrobial resistance in Y. enterocolitica should be regularly surveyed.     

Identification of substrates and chaperone from the Yersinia enterocolitica 1B Ysa type III secretion system

All pathogenic Yersinia enterocolitica strains carry the pYV plasmid encoding the Ysc-Yop type III secretion (TTS) system, which operates at 37 degrees C. In addition, biovar 1B Y. enterocolitica strains possess a second, chromosomally encoded, TTS system called Ysa, which operates, at least in vitro, under low-temperature and high-salt (LTHS) conditions. Six open reading frames, sycB, yspB, yspC, yspD, yspA, and acpY, neighbor the ysa genes encoding the Ysa TTS apparatus. Here we show that YspA, YspB, YspC, and YspD are secreted by the Ysa TTS system under LTHS conditions. SycB is a chaperone for YspB and YspC and stabilizes YspB. YspB, YspC, and SycB share some similarity with TTS substrates and the chaperone encoded by the Mxi-Spa locus of Shigella flexneri and SPI-1 of Salmonella enterica. In addition, Ysa also secretes the pYV-encoded YopE under LTHS conditions, indicating that YopE is a potential effector of both Y. enterocolitica TTS systems. YspC could also be secreted by S. flexneri, but no functional complementation of ipaC was observed, which indicates that despite their similarity the Ysa and the Mxi-Spa systems are not interchangeable. When expressed from the yopE promoter, YspB and YspC could also be secreted via the Ysc injectisome. However, they could not form detectable pores in eukaryotic target cells and could not substitute for YopB and YopD for translocation of Yop effectors.     

Endogenous ligands of Toll-like receptors: implications for regulating inflammatory and immune responses

Toll-like receptors (TLRs) have a crucial role in regulating immunity against microbial agents. Recent studies indicate that these receptors might also have an important role in regulating responses to endogenous stimuli, such as necrotic cells, heat-shock proteins and extracellular matrix breakdown products. Specifically, TLR2 and TLR4 were shown to mediate expression of inflammatory genes and trigger dendritic-cell 'maturation' by these agents. These intriguing findings suggest that the ancient family of TLRs are involved in the recognition, not only of microbes, but also of endogenous harmful stimuli. However, potential complications associated with microbial contamination of endogenous agents and the specific nature of in vivo responses induced by these agents remain to be determined.     

Convenient agarose medium for simultaneous determination of the low-calcium response and Congo red binding by virulent strains of Yersinia enterocolitica.

A simple, efficient method for identification and differentiation of Yersinia enterocolitica containing virulent plasmid-bearing clones is described. The method is based on temperature of incubation and the low calcium response of the organism in an agarose Congo red medium.     

Development of a highly specific assay for rapid identification of pathogenic strains of Yersinia enterocolitica based on PCR amplification of the Yersinia heat-stable enterotoxin gene (yst).

The chromosomal gene yst, which encodes a heat-stable enterotoxin of Yersinia enterocolitica, is a useful diagnostic marker because it occurs only in invasive strains of this species. A homologous gene also occurs in some strains of Yersinia kristensenii. Sequence analysis of the yst genes from two different strains of Y. enterocolitica and from Y. kristensenii revealed a substantial number of mismatches at the 3' ends of the yst genes of the so-called American and European biotypes of Y. enterocolitica. Moreover, several mismatches and a deletion of 5 codons were found in the yst of Y. kristensenii. These findings were used to develop a PCR-based assay for yst of Y. enterocolitica which yielded a detectable product in as little as 50 min. The assay was 100% specific in terms of its ability to identify potentially pathogenic strains of Y. enterocolitica regardless of biotype or serotype. The PCR yielded an amplicon that was visible on agarose gel electrophoresis from as few as 100 CFU, or 10 CFU when the PCR was combined with dot blot hybridization with a digoxigenin-labeled oligonucleotide probe that corresponded to an internal sequence of yst. These results establish the value of the yst gene as a target for the identification of pathogenic bioserotypes of Y. enterocolitica and the usefulness of PCR for this purpose.     

Cross-reactions between Yersinia enterocolitica serogroup 0:3 and other serogroups of the same species, as well as thirty-four other bacterial species

Cross-reactions between antigens from Yersinia enterocolitica serogroup 0:3 and 5 other members of the same species as well as 34 other bacterial species were studied by means of quantitative immunoelectrophoretic methods. A sonicated Y. enterocolitica antigen preparation and corresponding purified rabbit antibodies were used in a reference system that presented 58 regularly visible immunoprecipitates. One antigen was identified as specific for the Y. enterocolitica 0:3 serogroup and two antigens for the species Y. enterocolitica. Y. enterocolitica antigens cross-reacted widely with antigens from other Enterobacteriaceae, but only a few cross-reactions were registered with Gram-negative bacteria outside the Enterobacteriaceae. A partial cross-reaction between all Gram-positive bacteria included in our study and two Y. enterocolitica antigens was demonstrated.     

Plasmid-mediated surface fibrillae of Yersinia pseudotuberculosis and Yersinia enterocolitica: relationship to the outer membrane protein YOP1 and possible importance for pathogenesis.

The cell surface properties of Yersinia pseudotuberculosis and Yersinia enterocolitica mutants, constructed by insertional inactivation of genes located on the 40- to 50-megadalton virulence plasmid, were examined. Electron microscopy revealed an absolute correlation between expression of four plasmid-dependent, temperature-inducible properties related to the bacterial surface: (i) a fibrillar matrix covering the outer membrane, (ii) outer membrane protein YOP1, (iii) spontaneous autoagglutination, and (iv) mannose-resistant hemagglutination of guinea pig erythrocytes. Immunoelectron microscopy indicated that YOP1 is a structural component of the fibrillae. Experiments demonstrating inhibition of hemagglutination by anti-YOP1 monoclonal antibody suggested a potential role for YOP1 in adhesion. Insertional inactivation of the gene coding for YOP1, with resultant loss of the ability to express fibrillae, led to a significant reduction in the capacity of Y. enterocolitica, but not Y. pseudotuberculosis, to colonize the ileum of orogastrically infected mice. In both Y. enterocolitica and Y. pseudotuberculosis, inactivation of the genes coding for Ca2+ dependency reduced the ability to maintain intestinal colonization, regardless of the ability to express fibrillae. Both surface fibrillae and Ca2+ dependency seem to reflect pathogenic determinants which are required for the establishment of Y. enterocolitica infection. In Y. pseudotuberculosis, however, no clinical significance of the fibrillae has so far been defined.     

Identification of the Yersinia enterocolitica urease beta subunit as a target antigen for human synovial T lymphocytes in reactive arthritis.

The local T-cell response to bacterial antigens is involved in the pathogenesis of reactive arthritis (ReA). Here, we have identified a 19-kDa antigen of Yersinia enterocolitica O:9 recognized by Yersinia-specific synovial fluid CD4+ T cells in two patients with Yersinia-induced ReA. N-terminal amino acid sequencing of this protein revealed that it was identical to the 19-kDa urease beta subunit of Y. enterocolitica O:9. This protein has previously been shown to be arthritogenic in preimmunized rats after intra-articular injection. Analysis of the T-cell response to this protein showed that it contains several T-cell epitopes, one of which cross-reacts with other enterobacteria not able to induce ReA. This indicates that the arthritogenicity of the 19-kDa antigen is not a property of the 19-kDa protein alone but is dependent on its expression in bacteria able to induce ReA.     

HeLa cell infection by Yersinia enterocolitica: evidence for lack of intracellular multiplication and development of a new procedure for quantitative expression of infectivity.

The in vitro invasive properties of bacteria have frequently been studied by the use of HeLa cell cultures in chamber slides, using microscopic examination to enumerate intracellular bacteria. When this system was used to examine invasive properties of Yersinia enterocolitica, it resulted in rapid internalization of high numbers of bacteria during the infection phase which prevented subsequent discrimination of intracellular multiplication. A modified procedure was developed which standardized the ratio of bacteria to HeLa cells (i.e., multiplicity), the time for the infection phase, and the addition of specific antiserum with gentamicin for restricting bacterial uptake during the intracellular growth phase. Studies with this modified chamber slide system found that strains of human isolates of Y. enterocolitica (serotypes O:3, O:8, O:5,27, and O:6,30) exhibited different degrees of cell infection but did not multiply intracellularly. A second test system was developed that used roller tubes and viable cell counts for the enumeration of intracellular bacteria. This roller tube system confirmed that internalized bacteria did not multiply inside HeLa cells over a 24-h period. The roller tube system with viable cell counts for enumeration is a simplified technique for quantitative comparison of in vitro infectivity of HeLa cells by Y. enterocolitica.