Chromosomal-encoded siderophores are required for mouse virulence of enteropathogenic Yersinia species

Abstract Yersinia enterocolitica and Y. pseudotuberculosis are enteropathogenic for humans. Essential virulence functions of these pathogens are determined by a 40-mDa plasmid. Plasmid-bearing Y. pseudotuberculosis strains and Y. enterocolitica strains of serotypes 0 : 8, 0 : 13, 0 : 20 and 0 : 40 are lethal for mice. In contrast, human pathogenic Y. enterocolitica strains of serotype 0 : 3, 0 : 9 and 0 : 5.27 are not mouse-lethal. Using a sensitive siderophore-indicator CAS-agar, we were able to detect siderophore production in all mouse-lethal Y. enterocolitica and Y. pseudotuberculosis strains mentioned above. By Tn5-transposon insertions into the chromosome of a serotype 0 : 8 strain we obtained two siderophore-deficient mutants. Introduction of the virulence plasmid did not render these mutants mouse-lethal, indicating that siderophore production is an essential virulence factor. The human nonpathogenic, aerobactin-producing strains of Y. intermedia, Y. kristensenii and Y. frederiksenii remained avirulent for mice after receiving the virulence plasmid of Y. enterocolitica . Obviously the siderophore aerobactin does not contribute to virulence in the genus Yersinia .     

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.     

Detection of virulence markers in clinical strains of Yersinia]

The dispersion of plasmid pYV associated virulence markers in 474 Yersinia strains isolated from people has been studied. The ability to autoagglutination, calcium dependence of growth and the specific antigens were identified in 157 strains of traditionally pathogenic Yersinia enterocolitica serovars 03, 09, Yersinia pseudotuberculosis serovar I. They were not found in 223 strains of other 12 serovars of Yersinia enterocolitica, in 40 strains of Yersinia frederiksenii, Yersinia kristensenii, Yersinia intermedia. The proportion of virulent clones in the population of Yersinia is noted to depend on the conditions of its existence in vivo or in vitro. Identification of virulence markers is acknowledged to be expedient in epidemiological and ethiological estimation of the role of isolated Yersinia strains.     

Conjugative R-plasmid resistance of the causative agents of Yersinia infection

Nature, structure, occurrence and drug resistance of 160 strains of Y. pseudotuberculosis and 60 strains of Y. enterocolitica isolated from various sources within 1986-1988 were studied. In the strains of Y. pseudotuberculosis, the cell composition with respect to the requirements in calcium ions as well as the plasmid profiles with determination of the molecular weights of the plasmids in the antibiotic sensitive and resistant pathogens and R(+)-transconjugants were investigated. Some molecular genetic properties of the Yersinia R plasmids were also investigated. Antibiotic polyresistant strains of Y. enterocolitica were the most frequent donors of the R plasmids while the strains of Y. pseudotuberculosis were less frequently the donors, in the resistance pattern of which there were more frequent streptomycin and tetracycline resistance determinants. The conjugative R plasmids of Y. pseudotuberculosis were characterized by strict control of replication, repressed frequency of transfers, and a molecular weight of about 47 MD. Their replicones as a rule contained streptomycin and tetracycline markers determining resistance to streptomycin and tetracycline at the levels of 1250 and 156 micrograms/ml, respectively.     

Immunoelectrophoretic analysis of the antigenic composition of Yersinia

The antigenic composition of 24. Y. pseudotuberculosis newly isolated and reference strains, 7 Y. enterocolitica strains, as well as Y. pestis vaccine strain EV, has been studied by the method of immunoelectrophoresis in agar. The antigenic composition of these bacteria has been found to be complicated and to comprise not less than 8-11 antigens, and among them nonspecific protein antigens common for enterobacteria, the common generic antigen, the antigen common with Y. pestis, as well as O-antigens specific for each serovar are identified. Immunoelectrophoretic study has shown the possibility of Y. pseudotuberculosis O-antigen, serovar I, with Salmonella sera, serogroup A, and Y. enterocolitica 09 with brucellar and cholera sera.     

Comparative evaluation of Yersinia interaction with HEp-2 cells

In experiments on HEp-2 cells, the comparative characterization of the mechanism of invasion and the cytotoxic action of a number of Y. pseudotuberculosis strains, serovar 1, and Y. enterocolitica strains 03 and 09 (24 strains newly isolated from human patients and rodents and 5 laboratory strains at different degrees of attenuation) has been made on the basis of data obtained by optical and electron microscopy, as well as by cytoenzymological analysis. As revealed in these experiments, the invasion of Yersinia into epithelial cells and their capacity for intracellular multiplication depend on the cytotoxicity of the bacteria, most pronounced in Y. pseudotuberculosis, serotype 1, considerably less pronounced in Y. enterocolitica 09, and poorly pronounced (or absent) in Y. enterocolitica 03. Cytopathic changes are manifested by vacuolization, the exocytosis (clasmatosis) of the peripheral cytoplasm, impoverishment in organellae, the formation of autophagosomes, the shriveling of the nuclei and the perinuclear cytoplasm, the rounding of the cells and their separation from the glass surface. The development of these changes is accompanied by the increased activity of phosphatases diffusing into the cytoplasm and by the inhibition of cell respiration and dehydrogenase activity.     

The cultural properties and virulence of Yersinia]

Study of the cultivation properties of 82 enterobacterial strains has revealed that the colonies of virulent Y. enterocolitica (serovars O3, O9) and Y. pseudotuberculosis (serovar I) are temperature-sensitive. This sign, closely connected with the presence and expression of the virulence plasmid with a molecular weight of 44-48 MD, is not characteristic of other strains. Virulent Yersinia grown in nutrient agar for 48 hours at 37 degrees C form colonies which are smaller in diameter than those formed during cultivation at 26 degrees C (with the significance of differences t greater than or equal to 4), their diameter at 37 degrees C not exceeding 1.0 mm. The test for the determination of the temperature-sensitive morphology of Yersinia colonies, along with the tests for other virulence markers, is probably suitable for the detection of the causative agents of yersiniosis or pseudotuberculosis.     

Evaluation of usefulness for selected virulence markers for identifying pathogenic Yersinia enterocolitica strains. IV. Genes myfA and ureC

We check by polymerase chain reaction (PCR) the presence of gene ureC and myfA, encoding subunits of urease and Myf fimbriae, among clinical and food-originated strains of Yersinia to determine their usefulness as molecular virulence markers of Y. enterocolitica. The examinations were done on 130 clinical strains of Y. enterocolitica O:3/4 isolated in Poland from humans. All strains were obtained from stool and possessed the virulence plasmid pYV. In addition 40 isogenic, plasmid-cured strains were tested. The 52 strains including Y. enterocolitica (biotype 1A, 4, 2 and 1B), Y. pseudotuberculosis, Y. intermedia, Y. frederiksenii, Y. kristensenii, E. coli, Citrobatcer, Shigella and Salmonella were used as controls. The PCR assay resulted in detection of genes: ureC and myfA in genomic DNA of all 130 tested clinical strains of Y. enterocolitica pYV+, as well as in plasmid cured strains. Furthermore, ureC was found in all tested strains of Y. enterocolitica biotype A1 and in one strain of Y. intermedia and Y. kristensenii. In contrast to ureC, myfA was detected only in strains of Y. enterocolitica considered as pathogenic. Obtained results show, gene myfA seems to be the reliable virulence marker of Y. enterocolitica, whereas ureC is not recommended for identification of pathogenic strains of this species.     

Action of sodium chloride on Yersinia pseudotuberculosis and Yersinia enterocolitica

The bacteriostatic and bactericidal action of sodium chloride on 60 Y. pseudotuberculosis strains, 75 Y. enterocolitica strains and 158 urine-fermenting strains has been studied. A new specific feature of Y. pseudotuberculosis has been revealed: high sensitivity to sodium chloride. The suitability of the sodium chloride test has been shown for the identification of Yersinia and the differentiation of Y. pseudotuberculosis and Y. enterocolitica.     

A heat-modifiable outer membrane protein carries an antigen specific for the species Yersinia enterocolitica and Yersinia pseudotuberculosis

The outer membranes of gram-negative bacteria are considered to be of importance in host-bacteria interaction, in protective immunity, and occasionally in subclassification within a species. In this study, the outer membranes of several strains of Yersinia enterocolitica and Y. pseudotuberculosis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the appearance of the major proteins depended on the temperature at which they were solubilized in SDS. A protein was identified with the use of two-dimensional gels and preparative SDS-PAGE, which was equivalent to the "heat-modifiable protein" (protein II) of other Enterobacteriaceae species. A monoclonal antibody, 4G1, was generated against an isolated preparation of this Y. enterocolitica protein. This antibody was tested with whole cell bacterial antigens of 46 individual bacterial strains. The reactive strains included only Y. enterocolitica and Y. pseudotuberculosis. In addition, the reactivity of the 4G1 monoclonal antibody preparation could be absorbed only with Y. enterocolitica and Y. pseudotuberculosis, and not with other strains of bacteria. The reactivity of this 4G1 monoclonal antibody was also tested by the Western Blot technique. Six individual strains were tested: a Y. enterocolitica serotype 0:3, a Y. enterocolitica serotype 0:9, an Escherichia coli, a Salmonella typhimurium, a Shigella flexneri, and a Klebsiella pneumoniae. The 4G1 antibody reacted with only the proteins of the two Y. enterocolitica strains. In conclusion, the equivalent of the heat-modifiable protein was present in Y. enterocolitica and Y. pseudotuberculosis. Moreover, this protein also carried a species-specific antigenic determinant.     

The lytic activity of Yersinia pestis phage P 3d serovar

The lytic activity of plague phage II, serovar 3, with respect to 1,800 bacterial strains has been studied: 760 Yersinia pestis strains, 262 Y. pseudotuberculosis strains, 252 Y. enterocolitica strains, 166 Escherichia coli strains, 90 Shigella strains and 270 strains of other species. The phage has been found to lyse 81.8% of Y. pestis strains, 1 Y. pseudotuberculosis strain and 1 Y. enterocolitica strain. The representatives of other 19 bacterial species have proved to be resistant to the phage. Though having a wide range of action within Y. pestis, the phage does not lyse most of the strains of the causative agent of plague, isolated in certain natural foci. This fact offers promise for using the phage for the differentiation of Y. pestis.     

Studies on the pathogenicity of Yersinia enterocolitica. III. Comparative studies between Y. enterocolitica and Y. pseudotuberculosis

Comparative studies on pathogenicity between Yersinia enterocolitica and Yersinia pseudotuberculosis were performed using experimental infection systems in vivo and in vitro. All of thestra ins of both species successfully produced experimental enterocolitis in rabbits although the severity varied with the strains challenged. The changes were characterized by granulomatous lesions with necrobiotic centers in reticuloendothelial tissues of the intestine, mesenteric lymph nodes, liver and spleen. These strains uniformly had the ability to penetrate HeLa cells and to survive or multiply within cultured rabbit peritoneal macrophages. In addition, in infections with strain TP-2 or PST-III of Y. pseudotuberculosis, catarrhal inflammation all over the small intestine and/or focal necrosis and parenchymatous degeneration in the liver were observed, along with the granulomatous lesions. These strains, at the same time, exhibited cytotoxic effects on the cultured cells. The pathogenic factors of Y. enterocolitica are discussed in comparison with those of Y. pseudotuberculosis.     

Fatty acid composition of lipopolysaccharides of the strains of different species of Yersinia

The fatty acid composition of lipopolysaccharides of the strains of Y. enterocolitica, Y. intermedia, Y. frederiksenii and Y. ruckeri studied during cultivation on meat-peptone agar is characterized by the predominance of 3-hydroxytetradecanoic and dodecanoic acids. Closely related to the mentioned bacteria is the strain of Y. kristensenii which is distinguished only by its higher level of hexadecanoic acid. The strains of Y. pseudotuberculosis and the vaccine strain of Y. pestis have a uniform fatty acid composition of lipopolysaccharides with predominance of 3-hydroxytetradecanoic acid. Their relatively low level of dodecanoic acid conditions the characteristic fatty acid spectrum of lipopolysaccharides which differs from that of the above mentioned group of Yersinia. The peculiarities of the fatty acid composition of lipopolysaccharides of both groups of Yersinia are preserved during growth on meat-peptone broth, but the increase in the level of hexadecanoic acid balances the differences between Y. kristensenii, the other Y. enterocolitica-like bacteria and Y. ruckeri. The obtained results confirm close relationship of Y. pseudotuberculosis and Y. pestis, and also of Y. enterocolitica and Y. enterocolitica-like bacteria, showing propinquity of Y. ruckeri to the latter.     

Yersinia enterocolitica 03 findings on porcine tongues in comparison with yersiniosis incidence in man in Czechoslovakia

Positive isolations of Yersinia obtained in repeated bacteriological examinations of porcine tongues at three slaughter-houses in Prague and a single examination at the slaughter-house at Kladno were compared with notified yersiniosis morbidity. The incidence of illnesses caused by Y. enterocolitica 03 does not exceed values of 4.5/100,000 and 3.5/100,000 population in the Czech and Slovak Socialist Republics, respectively, and is equal to a sixtieth part of the notified shigellosis and salmonellosis morbidity. Cultivation of 334 pooled samples consisting of 1142 porcine tongues yielded 12 strains (1.05%) of Y. enterocolitica 03, five strains (0.44%) of Y. pseudotuberculosis and 55 strains (4.82%) of other Yersinia organisms (indole-positive serotypes). Because of the low isolation rates obtained for the individual Yersinia species, Y. enterocolitica 03 in particular, the isolation efficiency of different cultivation techniques and culture media was statistically evaluated for all Yersinia organisms jointly. Primary cultivation on deoxycholate-citrate medium yielded five of the 12 Y. enterocolitica 03 strains isolated. The other Yersinia strains grew only after preliminary propagation. Yersinia pseudotuberculosis grew almost exclusively (4 out of 5 strains) on McConkey's agar.     

The high-pathogenicity island of Yersinia pseudotuberculosis can be inserted into any of the three chromosomal asn tRNA genes

Pathogenicity islands (PAIs) have been identified in several bacterial species. A PAI called high-pathogenicity island (HPI) and carrying genes involved in iron acquisition (yersiniabactin system) has been previously identified in Yersinia enterocolitica and Yersinia pestis . In this study, the HPI of the third species of Yersinia pathogenic for humans, Y. pseudotuberculosis , has been characterized. We demonstrate that the HPI of strain IP32637 has a physical and genetic map identical to that of Y. pestis . A gene homologous to the bacteriophage P4 integrase gene is located downstream of the asn tRNA locus that borders the HPI of strain IP32637. This int gene is at the same position on the HPI of all three pathogenic Yersinia species. However, in contrast to Y. pestis 6/69, the HPI of Y. pseudotuberculosis IP32637 is not invariably adjacent to the pigmentation segment and can be inserted at a distance ≥ 190 kb from this segment. Also, in contrast to Y. pestis and Y. enterocolitica , the HPI of Y. pseudotuberculosis IP32637 can precisely excise from the chromosome, and, strikingly, it can be found inserted in any of the three asn tRNA loci present on the chromosome of this species, one of which is adjacent to the pigmentation segment. The pigmentation segment, which is present in Y. pestis but not in Y. enterocolitica , is also present and well conserved in all strains of Y. pseudotuberculosis studied. In contrast, the presence and size of the HPIs vary depending on the serotype of the strain: an entire HPI is found in strains of serotypes I only, a HPI with a 9 kb truncation in its left-hand part that carries the IS 100 sequence and the psn and ybtE genes characterizes the strains of serotype III, and no HPI is found in strains of serotypes II, IV and V.     

Evidence for two evolutionary lineages of highly pathogenic Yersinia species.

Sensitivity to Yersinia pestis bacteriocin pesticin correlates with the existence of two groups of human pathogenic yersiniae, mouse lethal and mouse nonlethal. The presence of the outer membrane pesticin receptor (FyuA) in mouse-lethal yersiniae is a prerequisite for pesticin sensitivity. Genes that code for FyuA (fyuA) were identified and sequenced from pesticin-sensitive bacteria, including Y. enterocolitica biotype 1B (serotypes O8; O13, O20, and O21), Y. pseudotuberculosis serotype O1, Y. pestis, two known pesticin-sensitive Escherichia coli isolates (E. coli Phi and E. coli CA42), and two newly discovered pesticin-sensitive isolates, E. coli K49 and K235. A 2,318-bp fyuA sequence was shown to be highly conserved in all pesticin-sensitive bacteria, including E. coli strains (DNA sequence homology was 98.5 to 99.9%). The same degree of DNA homology (97.8 to 100%) was established for the sequenced 276-bp fragment of the irp2 gene that encodes high-molecular-weight protein 2, which is also thought to be involved in the expression of virulence by Yersinia species. Highly conserved irp2 was also found in all pesticin-sensitive E. coli strains. On the basis of the fyuA and irp2 sequence homologies, two evolutionary groups of highly pathogenic Yersinia species can be established. One group includes Y. enterocolitica biotype 1B strains, while the second includes Y. pestis, Y. pseudotuberculosis serotype O1, and irp2-positive Y. pseudotuberculosis serotype O3 strains. E. coli Phi, CA42, K49, and K235 belong to the second group. The possible proximity of these two iron-regulated genes (fyuA and irp2), as well as their high levels of sequence conservation and similar G+C contents (56.2 and 59.8 mol%), leads to the assumption that these two genes may represent part of an unstable pathogenicity island that has been acquired by pesticin-sensitive bacteria as a result of a horizontal transfer.     

Lack of correlation between the presence of plasmids and fimbriae in Yersinia enterocolitica and Yersinia pseudotuberculosis

S kurnik , M. 1984. Lack of correlation between the presence of plasmids and fimbriae in Yersinia enterocolitica and Yersinia pseudotuberculosis. Journal of Applied Bacteriology 56 , 355–363.
Thirty-nine strains of Yersinia enterocolitica and 10 strains of Yersinia pseudoluber-culosis were studied for the presence of fimbriae, plasmids and two plasmid associated phenotypic expressions, autoagglutination and Ca²⁺ dependent growth at 37C. All of the strains studied became fimbriated, which was confirmed by electron microscopy and haemagglutination tests. Fimbriation was not correlated with the presence or absence of plasmids.     

The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis

The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.     

Coordinate involvement of invasin and Yop proteins in a Yersinia pseudotuberculosis-specific class I-restricted cytotoxic T cell-mediated response

Yersinia pseudotuberculosis is a pathogenic enteric bacteria that evades host cellular immune response and resides extracellularly in vivo. Nevertheless, an important contribution of T cells to defense against Yersinia has been previously established. In this study we demonstrate that Lewis rats infected with virulent strains of Y. pseudotuberculosis, mount a Yersinia-specific, RT1-A-restricted, CD8+ T cell-mediated, cytotoxic response. Sensitization of lymphoblast target cells for cytolysis by Yersinia-specific CTLs required their incubation with live Yersinia and was independent of endocytosis. Although fully virulent Yersinia did not invade those cells, they attached to their surface. In contrast, invasin-deficient strain failed to bind to blast targets or to sensitize them for cytolysis. Furthermore, an intact virulence plasmid was an absolute requirement for Yersinia to sensitize blast targets for cytolysis. Using a series of Y. pseudotuberculosis mutants selectively deficient in virulence plasmid-encoded proteins, we found no evidence for a specific role played by YadA, YopH, YpkA, or YopJ in the sensitization process of blast targets. In contrast, mutations suppressing YopB, YopD, or YopE expression abolished the capacity of Yersinia to sensitize blast targets. These results are consistent with a model in which extracellular Yersinia bound to lymphoblast targets via invasin translocate inside eukaryotic cytosol YopE, which is presented in a class I-restricted fashion to CD8+ cytotoxic T cells. This system could represent a more general mechanism by which bacteria harboring a host cell contact-dependent or type III secretion apparatus trigger a class I-restricted CD8+ T cell response.     

Evaluation of the usefulness of selected virulence markers for identification of virulent Yersinia enterocolitica strains. II. Genotypic markers associated with the pYV plasmid

Pathogenic strains of Yersinia enterocolitica bear virulence associated plasmid pYV. Unfortunately plasmid pYV is easily lost by these bacteria incubated at elevated temperatures (37 degrees C) or long stored at room temperatures. This sometimes makes difficult the detection of the virulence plasmid, especially by its isolation or biochemical tests. On the other hand, observations done by some authors suggest that polymerase chain reaction (PCR) could be useful for demonstration of the pYV plasmid of Yersinia strains. Accordingly to this observation the aim of the presented study was to check the usefulness of plasmid-localised genes virF and yadA, detected by PCR, for the identification of the virulent strains of Y. enterocolitica. In the presented study one hundred and fifty two clinical strains of Y. enterocolitica belonging to serogroup O3 were investigated by the PCR for the presence of genes virF and yadA. Bacterial strains were first tested for the presence of pYV plasmid. In addition the phenotypic features: calcium dependence, Congo red binding and autoagglutination were determined. In this way the virulence plasmid was found in 130 of 152 examined strains. For PCR studies also forty plasmid-cured strains of Y. enterocolitica and 32 non-Y. enterocolitica, Enterobacteriaceae strains were included. The obtained results show that the tested genes were present only in Yersinia strains possessing the pYV plasmid and no one non-specific PCR product was observed. The detection level of these genes in nested PCR permits to detect pathogenic Y. enterocolitica in suspension composed of 1 x 10(3) CFU/ml of pYV+ bacilli and 3 x 10(9) CFU/ml plasmid-cured, isogenic bacteria. In the study it was shown that genes virF and yadA were useful virulence markers, which could be helpful in clinical studies for the detection of the virulence plasmid in Y. enterocolitica strains long stored or incubated at elevated temperatures.