Plague virulence antigens from Yersinia enterocolitica.

The virulence of Yersinia enterocolitica, biotype 2, serotype O:8, in mice is related to its ability to produce plague V and W antigens. V and W antigens in Y. enterocolitica are shown to be immunologically identical to the previously described V and W antigens of Yersinia pestis and Yersinia pseudotuberculosis.     

Genetically manipulated virulence of Yersinia enterocolitica.

Mobilizable virulence plasmids of Yersinia enterocolitica of serotypes O:3 and O:9 were constructed by cointegration of a mobilizable vector into the virulence plasmids. The obtained cointegrates were mobilized into plasmidless Y. enterocolitica strains of serotypes O:3, O:5, O:8, and O:9. The transfer experiments revealed the existence of two different subgroups of plasmid-associated traits. (i) Animal virulence functions (mouse lethality and conjuctivitis provocation) were only transferable to plasmid-cured derivatives of virulent parent strains (serotypes O:3, O:8, and O:9), but they were not transferable to Y. enterocolitica antigen reference strains (serotypes O:3 and O:8) or to a plasmidless clinical isolate of serotype O:5. A further striking result was that a serotype O:8 strain regained the mouse lethality trait after receipt of a plasmid from a strain not lethal to mice. These results demonstrate that plasmid-mediated animal virulence functions are not uniformly expressed within Y. enterocolitica. (ii) The second subgroup of plasmid-mediated traits (calcium dependency, surface agglutinogens, HEp-2 cell adherence, and protein release) were transferable to all Y. enterocolitica recipient strains tested (serotypes O:3, O:5, O:8, and O:9 of different origin). For the first time HEp-2 cell adherence and temperature-induced release of five major protein species are described as transferable traits.     

Transfer of the virulence plasmid of Yersinia pestis to Yersinia pseudotuberculosis.

Transposon Tn5 insertion derivatives of the virulence plasmid pYV019 of Yersinia pestis were transferred by P1 transduction into a plasmid-free strain of Y. pseudotuberculosis. One of these plasmid derivatives conferred virulence upon the Y. pseudotuberculosis strain. This strain had the ability to express temperature-inducible plasmid-coded outer membrane proteins and was also found to be Ca2+ dependent.     

Cell adherence--a determinant of virulence in Yersinia enterocolitica

The adherence of pathogenic bacteria to eukaryotic cells plays a central role in their ability to colonize the mucosal epithelial surfaces. The adherence by Y. enterocolitica to the mucosal surface of the gut is the initiating event leading to penetration of mucosa. Adhesion of 10 probable pathogenic and one non-pathogenic isolate was studied using ileum and colon epithelial cells of mouse for 90 minutes. Adhesion study revealed that isolates of Y. enterocolitica had a good adhesive property while non pathogenic showed negligible adherence. All isolates showed better adherence to colon epithelial cells. The organism continued to be excreted in faeces up to 8-10 days after oral feeding. Adhesion positive isolates were found to be virulent when tested in mice for diarrhoea and death. Adhesion was found to be thermoregulated.     

The twin arginine translocation system is essential for virulence of Yersinia pseudotuberculosis

Yersinia species pathogenic to humans have been extensively characterized with respect to type III secretion and its essential role in virulence. This study concerns the twin arginine translocation (Tat) pathway utilized by gram-negative bacteria to secrete folded proteins across the bacterial inner membrane into the periplasmic compartment. We have shown that the Yersinia Tat system is functional and required for motility and contributes to acid resistance. A Yersinia pseudotuberculosis mutant strain with a disrupted Tat system (tatC) was, however, not affected in in vitro growth or more susceptible to high osmolarity, oxidative stress, or high temperature, nor was it impaired in type III secretion. Interestingly, the tatC mutant was severely attenuated via both the oral and intraperitoneal routes in the systemic mouse infection model and highly impaired in colonization of lymphoid organs like Peyer's patches and the spleen. Our work highlights that Tat secretion plays a key role in the virulence of Y. pseudotuberculosis.     

Serum resistance associated with virulence in Yersinia enterocolitica.

Yersinia enterocolitica strains that exhibited a calcium requirement for growth and autoagglutination at 37 degrees C were invariably virulent in rabbits, causing diarrhea and a high degree of lethality, and were capable of colonizing the intestinal lumen and establishing foci of infection on the Peyer's patches of mice. Strains that had lost the properties of calcium dependency and autoagglutinability were totally avirulent in rabbits and were quickly eliminated from the intestinal lumen and tissues of mice. Virulent and avirulent strains were shown to be equally invasive to HeLa cells. However, the virulent strains were resistant to the bactericidal action of normal serum, and this serum resistance was lost with the loss of virulence. Furthermore, the serum resistance of virulent strains was expressed, as were other properties, when strains were grown at 37 degrees C, but not at 27 degrees C. These results suggest that a virulence factor associated with serum resistance plays an essential role in the pathogenicity of Y. enterocolitica.     

Contribution of YopB to virulence of Yersinia enterocolitica.

The 70-kb virulence plasmid, pYV, of Yersinia enterocolitica encodes a number of secreted proteins (Yops) which are essential for virulence. YopD, the 33-kDa product of the lcrGVHyopBD operon, appears to be involved in delivering YopE and YopH (the Yersinia protein tyrosine phosphatase) into target cells. These proteins then act in concert to cause cytotoxicity in host cells. Previously, we reported that bacteria carrying transposon insertions in yopD are not cytotoxic for macrophages, show impaired tyrosine phosphatase activity in host cells, and are avirulent for mice (E. L. Hartland, S. P. Green, W. A. Phillips, and R. M. Robins-Browne, Infect. Immun. 62:4445-4453, 1994). trans complementation of yopD mutants of Y. enterocolitica with the yopD gene restores all these properties. In this study, we show that polar mutations in proximal genes of the lcrGVHyopBD operon also abrogated bacterial virulence and the capacity to induce cytotoxicity in mouse bone marrow-derived macrophages and HEp-2 epithelial cells. Moreover, trans complementation of a yopBD mutant with the yopD gene alone was not sufficient to restore the ability of the bacteria to cause cytotoxicity. Further work showed that YopB was required for cytotoxicity, dephosphorylation of host proteins, and virulence for mice. These findings indicate that YopB and YopD may serve a related function in Y. enterocolitica and that they may act together to deliver intracellularly acting Yops to their respective targets in host cells.     

Temperature-inducible surface fibrillae associated with the virulence plasmid of Yersinia enterocolitica and Yersinia pseudotuberculosis.

When cultivated at 37 degrees C in static broth, human clinical isolates of Yersinia enterocolitica (serogroups O:3, O:8, and O:9) and Yersinia pseudotuberculosis (serogroup O:III) produced numerous nonflagellar surface appendages, which appeared as a lawn of fine fibrillae, each having a diameter of 1.5 to 2.0 nm and a length of 50 to 70 nm. Cultivation at 22 degrees C resulted in complete disappearance of the fibrillae. The phenotypic expression of these appendages was correlated with the presence of the 40- to 48-megadalton virulence plasmid and was strongly affected by the growth medium. Evidence is presented which suggests that these plasmid-mediated, temperature-inducible surface fibrillae are responsible for autoagglutination and are related to production of one prominent, Sarkosyl-insoluble polypeptide of ca. 180 kilodaltons in the bacterial outer membrane.     

The influence of properties encoded by the Yersinia virulence plasmid on adhesion of Yersinia enterocolitica to ileal brush border membrane vesicles

The influence of plasmid-associated cell surface structures on the ability of Yersinia enterocolitica to bind to ileal brush border membrane vesicles (BBVs) was investigated. Rabbit or human BBVs were immobilized on polystyrene microtiter plates and adhesion of radiolabeled cells of Y. enterocolitica was determined. Strains of pathogenic serotypes carrying the Yersinia virulence plasmid (pYV+), as well as their isogenic plasmid cured derivatives (pYV-), adhered to immobilized BBVs, but adhesion of pYV+ organisms was markedly greater than that of pYV- ones. Strains belonging to non-pathogenic serotypes did not adhere significantly. The pYV+ strain Ye0301P+ did not express specific adhesion to glycolipids, nor was adhesion to BBVs reduced in the presence of various monosaccharides. Proteolytic digestion of surface structures on strain Ye0301P+ markedly reduced adhesion. pYV+ strains also demonstrated greater adhesion to a non-biological surface (polystyrene) and showed a higher degree of hydrophobicity than pYV- organisms as evaluated in a two-phase partitioning system. It is therefore likely that the plasmid-associated adhesion of Y. enterocolitica is promoted by one or more outer membrane proteins, that confer hydrophobicity to the bacterial surface.     

Superantigen YPMa exacerbates the virulence of Yersinia pseudotuberculosis in mice

Yersinia pseudotuberculosis, a gram-negative bacterium responsible for enteric and systemic infection in humans, produces a superantigenic toxin designated YPMa (Y. pseudotuberculosis-derived mitogen). To assess the role of YPMa in the pathogenesis of Y. pseudotuberculosis, we constructed a superantigen-deficient mutant and compared its virulence in a mouse model of infection to the virulence of the wild-type strain. Determination of the survival rate after intravenous (i.v.) bacterial inoculation of OF1 mice clearly showed that inactivation of ypmA, encoding YPMa, reduced the virulence of Y. pseudotuberculosis. Mice infected i.v. with 10(4) and 10(5) wild-type bacteria died within 9 days, whereas mice infected with the ypmA mutant survived 12 and 3 days longer, respectively. This decreased virulence of the ypmA mutant strain was not due to an impaired colonization of the spleen, liver, or lungs. In contrast to i.v. challenge, bacterial inoculation by the intragastric (i.g.) route did not reveal any difference in virulence between wild-type Y. pseudotuberculosis and the ypmA mutant since the 50% lethal doses were identical for both strains. Moreover, inactivation of ypmA gene did not affect the bacterial growth of Y. pseudotuberculosis in Peyer's patches, mesenteric lymph nodes (MLNs), and spleen after oral infection. Histological studies of spleen, liver, lungs, heart, Peyer's patches, and MLNs after i.v. or i.g. challenge with the wild type or the ypmA mutant did not reveal any feature that can be specifically related to YPMa. Our data show that the superantigenic toxin YPMa contributes to the virulence of Y. pseudotuberculosis in systemic infection in mice.     

Localization in Yersinia pestis of peptides associated with virulence.

An avirulent guanine auxotroph of wild-type Yersinia pestis was used to select isogenic mutants lacking invasive determinants of virulence including V and W antigens (Vwa-), genetically linked fibrinolysin, coagulase, and pesticin activities (Pst-), and the capacity to absorb exogenous pesticin and pigments including hemin (Pgm-). After growth in environments known to favor expression of these factors by the parent, cells were converted to spheroplasts and disrupted to obtain preparations of cytoplasm; particulate matter was separated into inner and outer membranes by sucrose gradient centrifugation. Peptides present in these fractions were then solubilized and compared by two-dimensional polyacrylamide gel electrophoresis. Components unique to Vwa+ cells, including V antigen, were restricted to the cytoplasmic fraction. In contrast, peptides possibly corresponding to fibrinolysin and coagulase were located primarily within the outer membrane of the Pst+ parent; pesticin was not identified. Similarly, a major outer membrane peptide, possibly representing the pesticin and pigment receptor, was peculiar to the Pgm+ parent. Accordingly, two of the virulence factors examined (Pst+ and Pgm+) can interact directly with host cells or fluids by virtue of their location on the bacterial surface. The remaining cytoplasmic Vwa+ determinant remains a candidate for a regulatory system whose role in pathogenicity is expression of functions required for intracellular survival.     

Characteristics of virulence in human isolates of Yersinia enterocolitica.

Yersinia enterocolitica serotypes O:3, O:8, O:9, O:5,27, O:4,32, and O:21 were virulent as determined by autoagglutination and calcium dependency at 35 degrees C and ability to produce guinea pig conjunctivitis and mouse diarrhea.     

Temperature-inducible outer membrane protein of Yersinia pseudotuberculosis and Yersinia enterocolitica is associated with the virulence plasmid.

A strain of Yersinia pseudotuberculosis which harbors a 63-kilobase plasmid was found to cause a lethal infection in Swiss albino mice. The rate of infection paralleled the ability of the pathogenic organism to attach to a monolayer of HeLa cells. One novel outer membrane protein (protein 1) with a molecular weight of 140,000 was found to be associated with the possession of the 63-kilobase plasmid not at 26 degrees C, and expression was moderately affected by the concentration of calcium in the growth medium. Moreover, it was found that synthesis of protein 1 associated outer membrane protein showing similar properties was also found to be expressed in plasmid-containing strains of Yersinia enterocolitica. The properties of protein 1 indicate that it could be identical to the previously described virulence W antigen.     

The rpoS gene from Yersinia enterocolitica and its influence on expression of virulence factors.

The chromosome of Yersinia enterocolitica encodes a heat-stable enterotoxin called Yst and a surface antigen called Myf, which closely resembles enterotoxin-associated fimbriae. Both factors could act in conjunction to produce diarrhea. Production of the enterotoxin is regulated by temperature, osmolarity, and pH and occurs only when bacteria reach the stationary phase. Myf production is regulated by temperature and pH and, as we show in this work, also occurs after the exponential growth phase. In an attempt to understand the late-phase expression of yst and myf, we cloned, sequenced, and mutagenized the gene encoding RpoS, an alternative sigma factor of the RNA polymerase involved in expression of stationary-phase genes in other enterobacteria. An intact rpoS gene was necessary for full expression of yst in the stationary phase but not for the expression of myf and of pYV-encoded virulence determinants.     

Genetic analysis of the formation of the Ysc-Yop translocation pore in macrophages by Yersinia enterocolitica: role of LcrV, YscF and YopN

The Ysc-Yop type III secretion (TTS) system allows extracellular Yersinia bacteria, adhering to eukaryotic target cells, to inject Yop effector proteins in the cytosol of these cells. The secretion apparatus, called the injectisome, ends up with a needle-like structure made of YscF. YopN, one of the proteins secreted by the injectisome is thought to act as a plug. YopB, YopD and LcrV, three other proteins secreted by the injectisome and called 'translocators' form a pore allowing translocation of the Yop effectors across the target cell plasma membrane. Here, we tested the role of LcrV, YscF and YopN in the formation of this pore in macrophages by monitoring the release of the low-molecular-weight fluorescent dye BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, 623Da) and of the high-molecular-weight lactate dehydrogenase (LDH, 135 kDa). BCECF is released through the translocation pore itself provided no Yop effector is trafficking through the channel. In contrast, LDH is released by the osmotic lysis of the target cell that occurs after pore formation. This release is reduced by the GAP activity of YopE. In order to study the role of LcrV, one has to circumvent the regulatory effect of LcrV on the synthesis of YopB and YopD. We observed here that this regulatory role of LcrV is lost in a yopQ mutant and hence we studied the role of LcrV in a yopQ mutant background. A lcrV, yopQ double mutant was deficient in pore formation while able to produce YopB and YopD. Pore formation was restored by the introduction of lcrV(+) but not yopQ(+) confirming that LcrV itself is directly required for pore formation. Bacteria secreting only YopB, YopD and LcrV could form pores, showing that YopB, YopD and LcrV are sufficient for pore formation provided they are secreted by the same bacterium. LcrV is not involved in secretion of YopB and YopD as suggested previously. Bacteria producing normal Ysc injectisomes, including the YscF needle but no translocators did not form pores, indicating that the needle is not sufficient by itself for pore formation, as was also suggested. yopN mutant bacteria formed needles and released BCECF even if they secreted the effectors. This observation suggests that many translocation pores are not filled in the absence of YopN and thus that YopN might form a link between the needle and the pore, guiding the effectors.     

Siderochrome production by Yersinia pestis and its relation to virulence.

P+ plague strains contained more siderochrome-producing organisms than P-. Siderochrome enhanced the mouse virulence of an F1+Vw+P1+P-Pu+ strains, inhibited P1 activity, and could be assayed by a paper disk titration method.     

The response regulator PhoP of Yersinia pseudotuberculosis is important for replication in macrophages and for virulence

Yersinia pestis and Yersinia pseudotuberculosis are closely related facultative intracellular pathogens. The response regulator PhoP was previously shown to be important for Y. pestis survival in macrophages and for virulence in a murine bubonic plague infection assay. Here the importance of PhoP for Y. pseudotuberculosis pathogenesis was investigated. Y. pseudotuberculosis phoP mutants were unable to replicate in low-Mg(2+) medium or in macrophages. phoP(+) Y. pseudotuberculosis strains initiated replication in macrophages after a lag period of approximately 5 h, as shown by fluorescence microscopy and viable count assays. Y. pseudotuberculosis phoP mutants died at a low rate in macrophages; there was no decrease in viability over the first 5 h of infection, and there was a 10-fold decrease in viability between 5 and 24 h of infection. Trafficking of phagosomes containing phoP(+) or phoP mutant Y. pseudotuberculosis was studied by using immunofluorescence microscopy and cathepsin D as a marker for lysosomes. Phagosomes containing phoP mutant Y. pseudotuberculosis acquired cathepsin D at a higher rate than phagosomes containing phoP(+) bacteria. However, the increased rate of marker acquisition for phagosomes containing mutant bacteria was only evident approximately 5 h after infection, suggesting that phoP mutants are able to retard phagosome maturation during the lag phase of intracellular growth. The results obtained with a Y. pestis phoP mutant were similar to those described above, except that the rates of intracellular killing and trafficking to cathepsin D-positive vacuoles were significantly higher. A Y. pseudotuberculosis phoP mutant was 100-fold less virulent than the wild-type strain in a murine intestinal infection model, suggesting that survival and replication in macrophages are important for Y. pseudotuberculosis pathogenesis.     

Evaluation of virulence factor testing and characteristics of pathogenicity in Yersinia enterocolitica

The virulence of 10 strains of Yersinia enterocolitica containing 42- to 44-megadalton plasmids (serogroups O:3; O:4,32; O:8; O:9; O:13,7; and O:21) was compared in mice and guinea pigs. Adult mice were more responsive than guinea pigs to the Sereny-like conjunctivitis test. In tests on suckling mice, all Yersinia strains harboring plasmids were lethal, whereas all strains without plasmids were nonlethal. All strains of serogroups O:4,32; O:8; O:13,7; or O:21 which harbored a plasmid gave positive results in the mouse Sereny, peroral, and intraperitoneal tests. A positive reaction in these tests was correlated with the ability of the strains to elaborate lipase. Because the Sereny, peroral, and intraperitoneal tests measured the same virulence factor(s), the findings in any of these three tests would accurately predict the results of the other two tests. Mice which survived the Sereny and peroral tests were subsequently challenged intraperitoneally with 1,000 mouse lethal doses of the virulent WA (O:8) strain. Those inoculated with plasmid-harboring strains were protected, whereas those inoculated with plasmid-free strains were not.     

In vitro assessment of virulence in Yersinia enterocolitica and related species.

We have examined 136 isolates of Yersinia species, comprising 112 strains of Yersinia enterocolitica, 12 of Y. frederiksenii, 8 of Y. intermedia, and 5 of Y. kristensenii, for the presence of 40- to 50-megadalton virulence-associated plasmids and expression of the following plasmid-associated characteristics: Congo red pigmentation (CR), calcium dependence, autoagglutination, hydrophobicity, resistance to normal human serum, and pathogenicity in mice. All 136 strains yielded both pigmented (CR+) and nonpigmented (CR-) variants. Only CR+ variants, however, were virulent for iron-overloaded, desferrioxamine B-treated mice (R. M. Robins-Browne and J. K. Prpic, Infect. Immun. 47:744-779, 1985). Although the in vitro virulence-associated characteristics generally occurred together, each one could be expressed independently. Strains of Y. frederiksenii, Y. intermedia, and Y. kristensenii also expressed individual virulence-associated properties. Of 53 Y. enterocolitica strains which were virulent for iron-overloaded, desferrioxamine-treated mice, all but one expressed every virulence-associated characteristic. Several strains which were avirulent for mice, however, demonstrated these characteristics in various combinations. Because many Yersinia strains, particularly environmental isolates, carried plasmids of 40 to 50 megadaltons, detection of plasmids provided little information about bacterial pathogenicity unless virulence-associated properties were also sought. The best in vitro predictor of virulence was autoagglutination, followed by calcium dependence. Because only CR+ variants expressed virulence-associated determinants, Congo red pigmentation is useful for selecting potentially virulent strains.