[Recent experimental studies with Pasteurella pseudotuberculosis (Yersinia pseudotuberculosis)].
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Reevaluation of the virulence phenotype of the inv yadA double mutants of Yersinia pseudotuberculosis.
Yersinia pseudotuberculosis and Yersinia enterocolitica are closely related human pathogens causing gastroenteritis. Invasin and YadA are two of the most extensively studied virulence factors of the Yersinia genus. Invasin is the primary invasion factor encoded by the inv gene on the chromosome and is required for the penetration of the epithelial cells. YadA is encoded by the yadA gene on the 70-kb virulence plasmid and has multiple functions. Previous studies indicate that an inv yadA double mutant of Y. enterocolitica is avirulent while an inv yadA mutant of Y. pseudotuberculosis is hypervirulent. In this study, we investigated this unexpected difference. New constructs of the inv yadA mutants of Y. pseudotuberculosis were made and tested in mice. These new constructs were not hypervirulent; rather, they maintained the same virulence as the wild-type strain. Further examination of the inv mutant used for the previous study revealed that it carries an aberrant inv phenotype and has an altered outer membrane profile and an altered colony morphology. Therefore, the mutants used previously were not isogenic to the parental wild-type strain, which may in part account for the difference in the results obtained.
Adhesins, Bacterial↗
The superantigen gene ypm is located in an unstable chromosomal locus of Yersinia pseudotuberculosis.
Yersinia pseudotuberculosis produces YPM (Y. pseudotuberculosis-derived mitogen), a superantigenic toxin that exacerbates the virulence of the bacterium in vivo. To date, three alleles of the superantigen gene (ypmA, ypmB, and ypmC) have been described. These genes are not found in all Y. pseudotuberculosis strains and have a low GC content, suggesting their location on mobile genetic elements. To elucidate this question, the genetic environment of the superantigen-encoding genes was characterized and 11 open reading frames (ORFs) were defined. Sequence analysis revealed that the ypm genes were not associated with plasmids, phages, transposons, or pathogenicity islands and that the superantigen genes were always located in the chromosome between ORF3 and ORF4. Nonsuperantigenic strains exhibited the same genetic organization of the locus but lacked the ypm gene between ORF3 and ORF4. A new insertion sequence, designated IS1398, which displays features of the Tn3 family, was characterized downstream of the ypmA and ypmC genes. A 13.3-kb region containing the ypm genes was not found in the genome of Y. pestis (CO92 and KIM 5 strains). We experimentally induced deletion of the ypm gene from a superantigen-expressing Y. pseudotuberculosis: using the association of aph(3')-IIIa and sacB genes, we demonstrated that when these reporter genes were present in the ypm locus, deletion of these genes was about 250 times more frequent than when they were located in another region of the Y. pseudotuberculosis chromosome. These results indicate that unlike other superantigenic toxin genes, the Yersinia ypm genes are not associated with mobile genetic elements but are inserted in an unstable locus of the genome.
Alleles↗
The ability to replicate in macrophages is conserved between Yersinia pestis and Yersinia pseudotuberculosis.
Yersinia pestis, the agent of plague, has arisen from a less virulent pathogen, Yersinia pseudotuberculosis, by a rapid evolutionary process. Although Y. pestis displays a large number of virulence phenotypes, it is not yet clear which of these phenotypes descended from Y. pseudotuberculosis and which were acquired independently. Y. pestis is known to replicate in macrophages, but there is no consensus in the literature on whether Y. pseudotuberculosis shares this property. We investigated whether the ability to replicate in macrophages is common to Y. pestis and Y. pseudotuberculosis or is a unique phenotype of Y. pestis. We also examined whether a chromosomal type III secretion system (TTSS) found in Y. pestis is present in Y. pseudotuberculosis and whether this system is important for replication of Yersinia in macrophages. A number of Y. pestis and Y. pseudotuberculosis strains of different biovars and serogroups, respectively, were tested for the ability to replicate in primary murine macrophages. Two Y. pestis strains (EV766 and KIM10(+)) and three Y. pseudotuberculosis strains (IP2790c, IP2515c, and IP2666c) were able to replicate in macrophages with similar efficiencies. Only one of six strains tested, the Y. pseudotuberculosis YPIII(p(-)) strain, was defective for intracellular replication. Thus, the ability to replicate in macrophages is conserved in Y. pestis and Y. pseudotuberculosis. Our results also indicate that a homologous TTSS is present on the chromosomes of Y. pestis and Y. pseudotuberculosis and that this secretion system is not required for replication of these bacteria in macrophages.
Animals↗
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.
Animals↗
Lack of correlation between haemagglutination and adherence to epithelial cells in Yersinia pseudotuberculosis.
Yersinia pseudotuberculosis was examined for its haemagglutinating activity and adherence to cultured epithelial cells (HEp-2) in relation to possession of a virulence (VW) plasmid and to growth conditions. VW-lacking (VW-) bacteria were isolated from ten VW+ strains of each serovar which, after they were grown on CFA plates at 37 degrees C, agglutinated the erythrocytes from five different species. In contrast to the bacteria possessing the plasmid (VW+) half of the VW- bacteria, grown on CFA plates at 37 degrees C, did not agglutinate any of the erythrocytes used and the other half agglutinated only human erythrocytes. Furthermore, when grown on CFA plates at 25 degrees C, neither VW+ nor VW- bacteria showed a haemagglutinating activity. When the bacteria were grown in CFA broth, only two strains grown at 25 degrees C did not agglutinate any of the erythrocytes tested. The VW+ and VW- bacteria of the remaining strains, grown either at 25 degrees C or 37 degrees C, showed relatively high haemagglutinating activity. Adherence to HEp-2 cells did not correlate with haemagglutinating activity in Y. pseudotuberculosis; the VW+ bacteria grown at 37 degrees C adhered to HEp-2 cells more efficiently than either the VW- derivatives or the VW+ bacteria grown at 25 degrees C, regardless of the growth medium. These results indicate that some of the haemagglutinins detected on Y. pseudotuberculosis are not involved in the adherence to HEp-2 cells.
Animals↗
Subcellular proteomic analysis of host-pathogen interactions using human monocytes exposed to Yersinia pestis and Yersinia pseudotuberculosis.
Yersinia pestis, the etiological agent of plague, is of concern to human health both from an infectious disease and a biodefense perspective. While Y. pestis and Yersinia pseudotuberculosis share more than 90% DNA homology, they have significantly different clinical manifestations. Plague is often fatal if untreated, yet Y. pseudotuberculosis causes severe intestinal distress but is rarely fatal. A better understanding of host response to these closely related pathogens may help explain the different mechanisms of virulence and pathogenesis that result in such different clinical outcomes. The aim of this study was to characterize host protein expression changes in human monocyte U937 cells after exposure to Y. pestis and Y. pseudotuberculosis. In order to gain global proteomic coverage of host response, proteins from cytoplasmic, nuclear and membrane fractions of host cells were studied by two-dimensional differential gel electrophoresis and relative protein expression differences were quantitated. Differentially expressed proteins, with at least 1.5-fold expression changes and p values of 0.01 or less, were identified by mass spectrometry including matrix-assisted laser desorption/ionization-MS or liquid chromatography tandem mass spectrometry. With these criteria, differential expression was detected in 16 human proteins after Y. pestis exposure and 13 human proteins after Y. pseudotuberculosis exposure, of which only two of the differentially expressed proteins identified were shared between the two exposures. Proteins identified in this study are reported to be involved in a wide spectrum of cellular functions and host defense mechanisms including apoptosis, cytoskeletal rearrangement, protein synthesis and degradation, DNA replication and transcription, metabolism, protein folding, and cell signaling. Notably, the differential expression patterns observed can distinguish the two pathogen exposures from each other and from unexposed host cells. The functions of the differentially expressed proteins identified provide insight on the different virulence and pathogenic mechanisms of Y. pestis and Y. pseudotuberculosis.
Electrophoresis, Gel, Two-Dimensional↗
Evidence for superantigen production by Yersinia pseudotuberculosis.
Yersinia pseudotuberculosis is an enteric pathogen that induces a variety of clinical symptoms, fever, scarlatiniform rash, diarrhea, vomiting, and arthritis. Characteristic histopathologic findings in Y. pseudotuberculosis infection such as lymphoid hyperplasia, typically seen in mesenteric lymph nodes, suggest that the stimulation of a large proportion of T lymphocytes may be involved in the pathogenesis of this infection. In this study, we assessed the mitogenic activity of culture supernatants of the clinical isolates of Y. pseudotuberculosis and investigated the mechanism by which these culture sups activate T cells. The culture sups, as well as partially purified fractions obtained by gel filtration, were found to selectively stimulate T cells bearing V beta 3, V beta 9, V beta 13.1, and V beta 13.2 compared with stimulation by anti-CD3. Furthermore, fibroblasts transfected with different HLA class II molecules, either HLA-DPw9, -DQw6, -DR1, or -DR4 Dw15, were capable of presenting Y. pseudotuberculosis culture supernatants to purified T cells. The T cell response to this sup was not restricted by donor HLA-DR types and was not neutralized by antibodies against the known staphylococcal superantigens, Staphylococcal enterotoxin (SE)A, SEB, SEC2, SED, SEE, and TSST1. These results suggest that Y. pseudotuberculosis produces superantigenic toxins that may mediate some of the systemic illnesses associated with infection by this organism.
Animals↗
Characterization of an amorphous and soluble hemagglutinin from Yersinia pseudotuberculosis.
Yersinia pseudotuberculosis which were screened out depending on auto-agglutination and Ca2+ dependency, were examined for their production of hemagglutinin (HA), and its purification and characterization were performed. The HA with a broad reactivity with various mammalian erythrocytes was recovered from the culture supernatant of these strains grown at 37 C but not 25 C. HAs from two strains, R148R and T1040, were purified by salt precipitation, gel filtration and anion-exchange chromatography by HPLC. Both purified HAs were cysteine-deficient acidic protein with an apparent molecular weight in the range of 15,000 to 16,000. N-terminal amino acid sequences of the first 25 residues were found to share 12% identity with that of afimbrial adhesin from enterotoxigenic Escherichia coli 2230. Immunoelectron microscopy and immunodiffusion test with polyclonal antiserum raised against the purified R148RHA demonstrated that the HA was associated with the amorphous aggregates which were detached from bacteria. These results suggest that the HA of Y. pseudotuberculosis belongs to a third type of HA produced by the yersinial species.
Amino Acid Sequence↗
Application of DNA microarrays to study the evolutionary genomics of Yersinia pestis and Yersinia pseudotuberculosis.
Yersinia pestis, the causative agent of plague, diverged from Yersinia pseudotuberculosis, an enteric pathogen, an estimated 1500-20,000 years ago. Genetic characterization of these closely related organisms represents a useful model to study the rapid emergence of bacterial pathogens that threaten mankind. To this end, we undertook genome-wide DNA microarray analysis of 22 strains of Y. pestis and 10 strains of Y. pseudotuberculosis of diverse origin. Eleven Y. pestis DNA loci were deemed absent or highly divergent in all strains of Y. pseudotuberculosis. Four were regions of phage origin, whereas the other seven included genes encoding a vitamin B12 receptor and the insect toxin sepC. Sixteen differences were identified between Y. pestis strains, with biovar Antiqua and Mediaevalis strains showing most divergence from the arrayed CO92 Orientalis strain. Fifty-eight Y. pestis regions were specific to a limited number of Y. pseudotuberculosis strains, including the high pathogenicity island, three putative autotransporters, and several possible insecticidal toxins and hemolysins. The O-antigen gene cluster and one of two possible flagellar operons had high levels of divergence between Y. pseudotuberculosis strains. This study reports chromosomal differences between species, biovars, serotypes, and strains of Y. pestis and Y. pseudotuberculosis that may relate to the evolution of these species in their respective niches.
Adaptation, Physiological↗
[A new R-plasmid from Yersinia pseudotuberculosis].
Yersinia pseudotuberculosis strain 140-P isolated from soil in the Far East was found to harbour an R-plasmid different from the plasmids that had been isolated from the bacteria previously. A new R-plasmid pLD140 is conjugation proficient and codes for the cellular resistance to streptomycin, tetracycline and sulfonamides. The plasid belongs to incompatibility group IncP. Its restriction endonucleases BamHI and SalI profile is different from the ones of the plasmids belonging to the RP4 family.
Drug Resistance, Microbial↗
Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis.
Yersinia pestis, the causative agent of plague, is a highly uniform clone that diverged recently from the enteric pathogen Yersinia pseudotuberculosis. Despite their close genetic relationship, they differ radically in their pathogenicity and transmission. Here, we report the complete genomic sequence of Y. pseudotuberculosis IP32953 and its use for detailed genome comparisons with available Y. pestis sequences. Analyses of identified differences across a panel of Yersinia isolates from around the world reveal 32 Y. pestis chromosomal genes that, together with the two Y. pestis-specific plasmids, to our knowledge, represent the only new genetic material in Y. pestis acquired since the the divergence from Y. pseudotuberculosis. In contrast, 149 other pseudogenes (doubling the previous estimate) and 317 genes absent from Y. pestis were detected, indicating that as many as 13% of Y. pseudotuberculosis genes no longer function in Y. pestis. Extensive insertion sequence-mediated genome rearrangements and reductive evolution through massive gene loss, resulting in elimination and modification of preexisting gene expression pathways, appear to be more important than acquisition of genes in the evolution of Y. pestis. These results provide a sobering example of how a highly virulent epidemic clone can suddenly emerge from a less virulent, closely related progenitor.
Bacteriophages↗
Susceptibility of wild mice to Yersinia pseudotuberculosis and Yersinia enterocolitica.
Laboratory-born progeny and adult populations of old world woods mice (Apodemus speciosus) trapped in a certain area where Yersinia pseudotuberculosis was prevalent among wild animals, were challenged with Y. pseudotuberculosis and Yersinia enterocolitica to determine the ecology of Yersinia in wild mice. The laboratory-born progeny population was associated with infection with Y. pseudotuberculosis but not with Y. enterocolitica serotype O:3 and was found to be nearly 200 times more susceptible to infection with Y. pseudotuberculosis than were the adult populations. A. speciosus convalescing from infection with Y. pseudotuberculosis showed protection upon subsequent challenge with homogeneous and heterogeneous serotypes of Y. pseudotuberculosis. These data suggest that the prevalence of Y. pseudotuberculosis in wild mice may begin with the infection in the newborn and thus spread to the Y. pseudotuberculosis naive adult mice populations.
Age Factors↗
Plasmid-determined cytotoxicity in Yersinia pestis and Yersinia pseudotuberculosis.
Yersinia pestis KIM5 was found to be cytotoxic for the IC21 and P388D1 mouse macrophage cell lines, as well as for resident peritoneal macrophages from C57BL/6 mice. Affected cells phagocytosed KIM5 inefficiently, became spherical, detached readily from culture dishes, and retained 51Cr poorly. The cytotoxic effect was dependent on the presence of the 75-kilobase plasmid pCD1. Because this plasmid also encodes the low calcium response (LCR), three Mu d1 insertion mutants previously shown to be LCR- and of reduced virulence in mice were examined for cytotoxicity; all were found to be atoxic. The insertions in these mutants lie within three distinct LCR loci (lcrB, C, and D). Like LCR, cytotoxicity was expressed only at 37 degrees C. Unlike LCR, it was not influenced by Ca2+ concentration, indicating that the V and W antigens are probably not involved. Yersinia pseudotuberculosis was found to have a similar plasmid-dependent cytotoxicity. Thus, biological activity observed as cytotoxicity in vitro may well be a common feature contributing to virulence of the yersiniae.
Animals↗
Ovine abortion and stillbirth due to purulent placentitis caused by Yersinia pseudotuberculosis.
Yersinia pseudotuberculosis was isolated from an aborted placenta and stillborn lamb from a sheep flock having multiple abortions. Given intravenously, it caused elevated body temperatures and purulent placentitis in eight of nine ewes. Two ewes died following infection at 2.5 months of gestation. Two ewes infected at 3.5 months gestation aborted; three infected at four months gave birth to weak, premature, or moribund lambs. One ewe infected at 4.5 months gave birth to a healthy lamb. One lamb which died minutes after birth had focal necrotizing hepatitis, a lesion observed in a stillborn lamb during the original disease outbreak. Y. pseudotuberculosis was reisolated from endometrial, placental, and fetal lesions of experimentally infected animals.
Abortion, Veterinary↗
Growth of Yersinia pseudotuberculosis and Yersinia enterocolitica biotype 3B serotype O3 inhibited on cefsulodin-Irgasan-novobiocin agar.
A total of 169 strains of Yersinia spp. were analyzed for their ability to grow on two different kinds of cefsulodin-Irgasan-novobiocin (CIN) agar containing 15 or 4 micrograms of cefsulodin per ml, on salmonella-shigella agar, and on MacConkey agar. CIN media inhibited the growth of Yersinia pseudotuberculosis and Yersinia enterocolitica biotype 3B serotype O3 (3B/O3) but not the growth of the other Yersinia organisms used. Relative to growth on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) with 6% yeast extract, 48 and 44% of Y. pseudotuberculosis and Y. enterocolitica 3B/O3 strains, respectively, were inhibited on CIN I agar (low cefsulodin concentration), and 83 and 54%, respectively, were inhibited on CIN II agar (high cefsulodin concentration) after incubation for 24 h at 32 degrees C. The inhibition of Y. pseudotuberculosis growth was significantly more extensive on CIN II agar than on CIN I agar. The MICs of cefsulodin and novobiocin clearly indicated a higher susceptibility for Y. pseudotuberculosis than for the other Yersinia organisms at 32 degrees C. All Y. pseudotuberculosis strains were susceptible to cefsulodin at 15 micrograms/ml (the approximate concentration used in CIN II agar). Y. enterocolitica 3B/O3 strains were resistant to cefsulodin, Irgasan, and novobiocin at the concentrations used in CIN media. These findings show that cefsulodin inhibits the growth of Y. pseudotuberculosis at the concentration used in CIN media and that growth inhibition of Y. enterocolitica 3B/O3 is related to a component of the CIN Base.
Agar↗
Comparison of the ability of enteroinvasive Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Yersinia enterocolitica to enter and replicate within HEp-2 cells.
Salmonella typhimurium, enteroinvasive Escherichia coli, Yersinia pseudotuberculosis, and Yersinia enterocolitica possess the ability to enter intestinal epithelial cells. We used a quantitative tissue culture model employing HEp-2 cells to compare the abilities of these bacteria to enter epithelial cells. S. typhimurium and Yersinia species were highly infective for HEp-2 cells but were unable to replicate extensively intracellularly. Enteroinvasive E. coli exhibited low infectivity but replicated extensively intracellularly. The growth of enteroinvasive E. coli led to destruction of the HEp-2 monolayer, whereas Yersinia spp. and S. typhimurium were maintained intracellularly for prolonged periods without damage to the monolayer. The ability of enteroinvasive E. coli to enter HEp-2 cells required prior growth at 37 degrees C; neither S. typhimurium nor Yersinia spp. exhibited this temperature dependence for cell entry. An E. coli K-12 derivative containing a 230-kilobase plasmid from enteroinvasive E. coli was constructed. This derivative shared all the invasive characteristics of the parental enteroinvasive strain, suggesting that determinants required for cell entry and intracellular multiplication were at least partially plasmid encoded. An HB101 derivative containing a cloned invasion determinant from Y. pseudotuberculosis was constructed in our laboratory. HEp-2 monolayers were coinfected with these two K-12 derivatives to compare invasion determinants from enteroinvasive E. coli with those of Y. pseudotuberculosis in a common genetic background. Results from these experiments suggest that these organisms reside within separate intracellular compartments.
Cells, Cultured↗
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.
Agglutinins↗