Structural-functional characteristics of Yersinia pseudotuberculosis plasmid pVM82

The restriction map of Yersinia pseudotuberculosis plasmid pVM82 was established using the "chromosome walking" method. According to transpositional mutagenesis, the plasmid pVM82 appeared to be conjugative and was able to be transmitted from Y. pseudotuberculosis to the E. coli K-12 cells.     

Alternative localization of the determinant of pathogenicity coded by the Yersinia pseudotuberculosis pVM82 plasmid]

The chromosomal DNA regions in Yersinia pseudotuberculosis strains occur that are homologous to 25 Md DNA segment of the plasmid pVM82 encoding the bacterial capability of immunosuppression. The character of the chromosomal DNA regions dispersion reacting with the 25 Md segment probes is different in epidemiologically hazardous and nonvirulent strains of Yersinia pseudotuberculosis. The specific DNA regions occur as well as identical ones. The suppression of antibody formation to a number of main Yersinia pseudotuberculosis antigens by epidemiologically hazardous strain is demonstrated. The suppression is analogous to the one previously described for Yersinia pseudotuberculosis strains harbouring the plasmid pVM82.     

Effects of growth temperature and pVM82 plasmid on fatty acids of lipid A from Yersinia pseudotuberculosis

Effects of cultivation temperature (8 or 37 degrees C) and plasmid profile on the lipid A fatty acids of three isogenic Yersinia pseudotuberculosis strains (plasmidless (82-) and strains containing pVM82 (82+) or p57 (57+) plasmids) obtained by alkaline hydrolysis of the whole bacterial cells and differentiated from fatty acids of other membrane lipids were investigated. On the basis of the analysis, it is concluded that lipids A of all studied samples contain 3-hydroxytetradecanoic and dodecanoic acids, a part of which exists as the 3-dodecanoyloxytetradecanoic derivative. The effect of temperature appears in the higher contents of ester- and amide-linked 3-acyloxyalkanoic residues in lipid A from the "cold" variants of the bacteria and is determined by chromosomal genes. The plasmid effect is seen as various responses of the isogenic derivatives to change of growth temperature: in cells of strains 82+ and 82- grown in the cold, the share of lipid A fatty acids in the total population of cellular fatty acids is reduced, while in strains with plasmid p57 it is increased. The temperature variants of the 57+ strain differ by the low contents of amide-linked 3-acyloxyalkanoic acids. Finally, lack of plasmid pVM82 in the "warm" variants of the bacteria results in accumulation of glycolipid molecules deprived of dodecanoic acid. Correlation between growth temperature and plasmid profiles, on one hand, and lipid A fatty acid composition and potential pathogenic properties of the Y. pseudotuberculosis, on the other hand, and also possible mechanisms of thermal adaptation of this organism are discussed.     

Phenotypic features of a strain of Yersinia pseudotuberculosis with the pVM82 plasmid, detected at pseudotuberculosis outbreak foci]

The phenotypic properties conferred to Yersinia pseudotuberculosis cells by the genetical determinants of a 25Md fragment of the plasmid pVM82 coding for the modified cellular immune response in the infected organism. The fragment was shown to determine the conjugative properties of the plasmid, the resistance of bacterial cells to a number of hydrophobic agents and cellular ability to absorb the Congo red dye. The latter confirms the presence of additional structural components in the cell wall of the strain harbouring the plasmid pVM82. The increased resistance of the plasmid-containing strain to bactericidal effect of the blood plasma was demonstrated as compared with the resistance of the strains harbouring the p57 plasmid lacking the 25Md fragment or no plasmid at all.     

Synthesis of lipopolysaccharides in the bacterium Yersinia pseudotuberculosis: effect of the pVM82 plasmid and growth temperature

The composition and structure of lipopolysaccharides (LPS) of three isogenic strains of Yersinia pseudotuberculosis serovar O:1b (without plasmids (82-) and with plasmids pVM82 (82+) or p57 (57+)) grown at 8 or 37 degrees C were studied by chemical and immunochemical methods, SDS-polyacrylamide gel electrophoresis, and 13C-NMR spectroscopy. At the lower temperature, the (82-) and (82+) strains synthesized S-form of LPS with similar structure characterized by high acylation and immunochemical activity. On the other hand, LPS of the (82+) strain had shorter carbohydrate chains than LPS of the (82-) strain. The contents of LPS were decreased in cells of the plasmid-free strain grown at the higher temperature. LPS isolated from these cells were of the R-form and had low acylation and immunochemical activity. Total LPS content in cells of the (82+) strain did not significantly depend on the growth temperature. LPS of the warm variant of these bacteria contained a polysaccharide fragment and had moderate immunochemical activity. The cells of the (57+) strain at both growth temperatures had low LPS contents and produced LPS of low acylation without O-specific chains (cold variant) or containing O-polysaccharide with low polymerization degree (bacteria grown at 37 degrees C). The data indicate that in the absence of the plasmids, LPS synthesis is encoded by the chromosomal genes in pseudotuberculosis bacteria. Expression of the genes involved in LPS synthesis is regulated by the temperature of bacterial growth. Genes responsible for temperature-dependent regulation of LPS biosynthesis are located on chromosomal DNA. The pVM82 plasmid includes two gene groups; one group is localized in a 57-mD fragment of DNA and inhibits LPS synthesis, suppressing temperature-dependent regulation of the synthesis. The genes located in a 25-mD fragment of the pVM82 plasmid are de-repressors of the 57-mD fragment, and they restore the ability of pseudotuberculosis bacteria to synthesize relatively long LPS at both growth temperatures.     

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.     

Binding to collagen by Yersinia enterocolitica and Yersinia pseudotuberculosis: evidence for yopA-mediated and chromosomally encoded mechanisms.

Binding of Yersinia enterocolitica and Yersinia pseudotuberculosis strains to type I, II, and IV collagens has been studied. Wild-type strains which harbored the 40- to 50-megadalton virulence plasmid specifically bound all three types of collagen. Curing of the virulence plasmid or Tn5 insertion in the yopA gene encoding the temperature-inducible outer membrane protein YOP1 abolished the binding of all three collagen types to Y. enterocolitica and type I and II collagens to Y. pseudotuberculosis. Full binding capacity was restored by introduction of the yopA gene into nonbinding Yersinia strains. Binding of type I, II, and IV collagens was expressed in Escherichia coli constructs harboring the yopA gene of either Y. enterocolitica or Y. pseudotuberculosis. The interaction of bacterial cells with type I collagen could be blocked by nonradiolabeled native collagens or denatured collagen but not with other serum and connective-tissue proteins. Unlabeled collagen could not displace bound radiolabeled collagen. The binding was inhibited by YOP1-specific polyclonal antibodies, in contrast to normal rabbit serum. The interaction was rapid and was quite resistant to heat treatment, to proteolytic enzymes, to various pHs in both acidic and alkaline ranges, and to the chaotropic agent urea. We propose that this newly identified interaction may be involved both in the first steps of the pathogenesis and in the complications of Yersinia infections affecting connective tissue.     

Various characteristics of Yersinia pseudotuberculosis determined by plasmid 45 Md associated with calcium dependence

The clones of two types (T+ and T-) have been identified among the strains of Y. pseudotuberculosis. The difference in the morphology of the clones is based on the ability of T+ clones to agglutinate in the process of growth. The identified clones are different in calcium dependence, in the ability to agglutinate and in their virulence for the laboratory animals. The differences have been proved to be connected with the presence of a 45 Md plasmid in T+ cells. Replication of this plasmid is suppressed by the acridine orange dye in concentrations of 12.5 or 25.0 mkg X ml-1. The plasmid is spontaneously lost from the strains during their continuous storage. The microscopy of colonies permits the selection of clones with the parental phenotype from the populations having lost the 45 Md plasmid.     

Analysis of the plasmid composition of Yersinia pseudotuberculosis strains and its use for typing pseudotuberculosis pathogens

Electrophoresis in agarose gel has been used to study the plasmid spectra of 854 Yersinia pseudotuberculosis strains isolated from different sources. The plasmids found in the microbial strains are represented by the elements with molecular masses 82; 57; 45; 5.5; 4.4; 3.5; 2.7; 2.4; 2.3 Md. The variable spectra of plasmids is peculiar only for serovar I of Yersinia pseudotuberculosis. Plasmids p45 and p82 are classified as the main, while other plasmids as auxiliary ones. In accord with the classification all plasmid containing strains are divided into 8 plasmid strains. Using the proposed method for intraspecific typing of Yersinia pseudotuberculosis permits one to perfect the epidemiological analysis of pseudotuberculosis infection and make concrete the direction of prophylactic and antiepidemic measures.     

Expression of Yersinia pestis antigens encoded by the Ca2+-dependence plasmid

Antigens coded by the Ca2(+)-dependance plasmid were found in the cultural medium, cytoplasm and outer membranes of the three monoplasmid (pCadV) strains of Yersinia pestis with the different basic properties. The presence of 20 mM of Mg2+ at least in the medium is necessary for optimal expression of these proteins. The existence of strain differences in the bacterial cells reaction to temperature, cultivation medium has been demonstrated. No difference in the pCad-dependent proteins was found in Yersinia pestis and the causative agents of pseudotuberculosis, enterocolitis.     

A highly specific one-step PCR - assay for the rapid discrimination of enteropathogenic Yersinia enterocolitica from pathogenic Yersinia pseudotuberculosis and Yersinia pestis

Based on differences within the yopT-coding region of Yersinia. enterocolitica, Y pseudotuberculosis and Y pestis, a rapid and sensitive one-step polymerase chain reaction assay with high specificity for pathogenic Y enterocolitica was developed. By this method pathogenic isolates of Y enterocolitica can be easily identified and discriminated from other members of this genus. The entire coding sequence of the yopT effector gene of Y. pseudotuberculosis Y36 was determined.     

Effect of preparations of protein Yop encoded by Yersinia pestis calcium-dependence plasmid on mice

The impact of the preparations of Y. pestis secreted proteins Yop (YopH-M, YopB, YopD-N, YopE) on mice immunized with 3 s.c. injections was studied. Though these proteins failed to protect the animals from plague, they stimulated the immunobiological transformation in the immunized animals. YopB and YopD-N were found to have the highest immunobiological activity with respect to mice. The preparation of YopB induced the production of the highest titers of specific antibodies and stimulated cell-mediated immune response. The injection of YopD-N to mice led to a considerable decrease in the proliferative capacity of splenocytes in vitro in response to stimulation with nonspecific mitogen ConA, as well as to pathological changes in the kidneys.     

Specific proteolysis by fibrinolysin-coagulase from Yersinia pestis of Yersinia pseudotuberculosis outer membrane proteins coded by the Ca(2+)-dependence plasmid]

The pesticinogenicity 9.5 kb plasmid from Yersinia pestis strain EV76 has been marked by the kanamycin phosphotransferase gene inserted into PstI site and designated pP3. The obtained plasmid pP3 determines the synthesis of 45 kd pesticin, alpha and beta-forms of fibrinolysin coagulase (37 and 35 kd) and the 29, 19 and 13 kd proteins in Escherichia coli mini cells. When transferred into Yersinia pseudotuberculosis strain 6933 the plasmid causes the proteolysis of outer membrane proteins. The 150 kd protein is reduced to 138 kd, the 48.5 kd protein is reduced to 45 kd. The proteins secreted into the cultural medium (51 and 38 kd) are also cleaved. The proteolysis of the 150 kd protein was found to occur at the stage of secretion via the inner membrane. The purified fibrinolysin coagulase from Escherichia coli strain JM83 harbouring the plasmid pP3 induces the proteolysis in vitro of the isolated membrane proteins from Yersinia pseudotuberculosis strain 6953 similar to the proteolysis registered in vivo.     

Characterization of a Superantigen Produced by Yersinia pseudotuberculosis

Abstract

Yersinia species are Gram-negative coccobacilli consisting of three pathogenic species, Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, and five nonpathogenic species, Y. kristensenii, Y. frederiksenii, Y. intermedia, Y. rohdei, and Y. aldovae. The former three species are primary pathogens of wild and domestic animals and birds. In the human, Y. pestis causes plague, or black death, while Y. pseudotuberculosis and Y. enterocolitica produce milder forms of disease varying from diarrhea and abdominal pain to more systemic symptoms such as fever, scarlatiniform skin rash, conjunctivitis, erythema nodosum, and lymphadenopathy (1–3). Complications of reactive arthritis, acute uveitis, coronary aneurysms, and acute renal failure are not infrequently reported after the latter two Yersinia infections (4–8). The mechanisms by which these organisms mediate these complicated symptoms are poorly understood. However, the preferential avidity for lymphoid tissues seen in these species and the characteristic histopathological finding of lymphoid hyperplasia mainly seen in mesenteric lymph nodes (9–10) suggest that the stimulation of a large proportion of T lymphocytes may be involved in the pathogenesis of this infection.     

A new alpha-helical coiled coil protein encoded by the Salmonella typhimurium virulence plasmid.

A new protein of Salmonella typhimurium was identified and characterized. The gene (tlpA) encoding this protein (TlpA) was isolated from the large virulence-associated plasmid of S. typhimurium and sequenced in order to predict the primary structure of TlpA. tlpA encodes a 371-amino acid soluble protein with a calculated M(r) of 41600 and pI of 4.63. Secondary structure predictions and sequence statistics of TlpA indicated a predominant alpha-helical configuration and presence of heptapeptide repeat motifs characteristic of coiled coil proteins. Purified TlpA was shown to have biochemical properties similar to those of coiled coil proteins, including adoption of an alpha-helical configuration and a tendency to form homodimers. Furthermore, TlpA possessed heat resistance, evidence for a chain register and altered mobility in urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels which are characteristics of tropomyosins. TlpA shows 32% overall sequence similarity with rat cardiac myosin and 36% similarity with horse platelet beta-tropomyosin over 226 residues, whereas selected regions possessed significant sequence identities with myosins, tropomyosins, and alpha-helical surface proteins of Streptococcus pyogenes. Our results indicate that TlpA represents a new member of prokaryotic coiled coil proteins.     

A cloned chromosomal DNA fragment which differentiates Yersinia pestis from Yersinia pseudotuberculosis and Yersinia enterocolitica

Chromosomal DNA from reference Yersinia strains was digested individually with 9 restriction endonucleases. DNA fragments were separated and analyzed by electrophoresis through agarose gels. The clearest fragment patterns were obtained when EcoRI was employed. The Y. pestis fragment pattern obtained after the use of this enzyme showed the presence of a unique DNA fragment with molecular mass 1400 bp. This DNA fragment was cloned, purified, labeled with 32P and then used to probe EcoRI digests of all three Yersinia species. A strong hybridization signal was obtained with Y. pestis strain. No such signal was found with Y. pseudotuberculosis or Y. enterocolitica. These results indicate that the DNA fragment is species specific and could be used as a diagnostic DNA probe for Y. pestis.     

Production of thermolabile enterotoxins of Escherichia coli by Yersinia enterocolitica and Yersinia pseudotuberculosis

The plasmids pCG86 and RP4elt coding for thermolabile enterotoxins of Escherichia coli (LT) were transferred in conjugation to Yersinia enterocolitica and Yersinia pseudotuberculosis cells. Both plasmids were stably inherited by the recipient cells. The elt genes of the toxins were expressed in Yersinia cells at the level comparable to the one registered in Escherichia coli cells. In the broth cultures of transconjugant cells the major part of LT toxin is bound with cells (74-97%). The obtained data may serve an experimental basis in favour of possibility of Ent+ strains of Yersinia enterocolitica and Yersinia pseudotuberculosis formation in nature and expediency of search and diagnosing of such strains.     

A Yersinia pseudotuberculosis protein which cross-reacts with HLA-B27

The most-debated question in the investigation of the spondyloarthropathies has been whether there is molecular mimicry between host HLA-B27 antigens and the arthritis-causing pathogens. We have generated a monoclonal anti-HLA-B27 antibody in our laboratory and have used a radioimmunoassay to screen a panel of bacterial species. Two strains of Yersinia pseudotuberculosis were found to be highly reactive. The cross-reactive Yersinia component was identified by Western blot to be a 19,000 component. A preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis chromatography apparatus was constructed to isolate milligram quantities of this component. To verify that the component carried the HLA-B27-specific epitope, rabbits were hyperimmunized with the purified materials. Affinity-purified antibodies from one of the immunized rabbits indeed carried anti-HLA-B27 activity. Last, antibodies generated against synthetic peptides derived from the HLA-B27.1 amino acid sequence were tested against the Yersinia component. Positive reactivity was found with antibodies generated against a peptide spanning residues 69-83 of the HLA-B27.1 protein. Since this resides in the segment responsible for the allotypic specificity of the antigen, these experiments establish the presence of molecular mimicry to a high degree of confidence.