Studies on the interactions of immunostimulated macrophages and Yersinia enterocolitica O:8

Immunological and electron microscopy investigations of the phagocytic and killing activities of peritoneal macrophages from rats and mice against Yersinia enterocolitica serotype O:8 cells were performed. The effect of in vivo application of cytoplasmic membranes (CM) from the stable Escherichia coli WF+ L-form on macrophage activity was also studied. It was established that rat macrophages more actively phagocytosed the plasmidless pYV(-) Y. enterocolitica cells, compared to the plasmid-bearing pYV(+) Y. enterocolitica cells. The killing ability against both variants of the Y. enterocolitica strain was significantly enhanced in macrophages from CM-treated rats after 2 h, 4 h, and 24 h incubation. The CM treatment enhanced the phagocytic activity of the macrophages. The in vitro interaction of normal and immunostimulated rat macrophages with both pYV(+) and pYV(-) variants of Y. enterocolitica did not lead to any additional apoptotic and necrotic changes in macrophages compared to control macrophages, which were cultivated without Y. enterocolitica. Electron-microscopic investigation showed that mouse macrophages eliminated Y. enterocolitica pYV(+) cells in vivo after 24 h. No engulfed or digested bacterial cells were observed. Activation of cell surfaces and vacuolization of macrophage cytoplasm, both of CM-treated non-infected and infected mice, were observed. The experimental results showed that Y. enterocolitica pYV(+) cells could be eliminated by peritoneal macrophages.     

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.     

Evaluation of the usefulness of selected virulence markers for identification of virulent strains of Yersinia enterocolitica strains. III. Chromosome markers of virulence

It is well known that virulent strains of Y. enterocolitica bear the virulence-associated plasmid pYV. Moreover some authors consider that the pathogenic strains of these bacteria have chromosome encoded phenotypic and genotypic features such as: genes ail and yst which could be used as virulence markers. The virulent strains of Y. enterocolitica do not produce pyrasinamidase and are not able to ferment salicin and cannot hydrolyse esculin. In addition these strains produce thermostable enterotoxin called YST and protein Ail (attachment invasion locus). In contrast to phenotypic virulence makers the biological function of proteins Ail, YST and nucleotide sequence of genes ail and yst is well described. In the presented study one hundred thirty virulence plasmid bearing Y. enterocolitica strains belonging to serogroup O3 were examined for the presence of genes ail, yst, and were tested for their inability to pyrasinamidase production, salicin fermentation and esculin hydrolysis. In addition forty pYV plasmid-cured isogenic strains were included in to the study. Genes ail and yst were detected by polymerase chain reaction (PCR). The obtained results indicate that all tested 130 pYV+ Y. enterocolitica strains as well as 40 plasmid-cured isogenic strains have carried ail and yst genes. All tested strains did not produce pyrasinamidase, hydrolyse esculin and ferment salicin. This generally was in agreement with the observations done by other authors and suggest that the chromosomal virulence markers, especially well described genes ail and yst, could be useful for excluding the potential virulence of Y. enterocolitica strains, which had lost pYV plasmid and have no ail or yst genes. Therefore, in clinical studies, Y. enterocolitica strains isolated directly from patients should be primarily tested for the presence of the virulence plasmid and secondarily, the negative ones could be examined for the presence of the chromosomal virulence markers.     

Evaluation of the usefulness of selected virulence markers for the identification of virulent Yersinia enterocolitica strains. I. Phenotypic markders associated with Plasmid pYV

The species Yersinia enterocolitica includes either pathogenic or non-pathogenic strains. Therefore it is necessary to differentiate virulent bacilli from other. It is well known that pathogenic strains of Y. enterocolitica bearing virulence associated plasmid called pYV, which could be demonstrated by its isolation or detected by the presence of specific, phenotypic properties directly related with this plasmid. The aim of the presented paper was to check the ability of some phenotypic virulence markers associated with pYV, to detection of pathogenic Y. enterocolitica strains. In the presented work 152 (130 carrying pYV) clinical strains of Y. enterocolitica O3 isolated mainly from stool were examined for the presence of phenotypic virulence markers such as: calcium dependency, Congo-red binding, autoagglutination and agglutination with Mangifera indica extract. Both first features were detected parallel, on the same plate, using CRMOX (Congo-red, Magnesium Oxalate) agar. The detection of the tested markers in the examined strains was compared with the presence of virulence plasmid. The obtained results confirmed the observations done by other authors that Y. enterocolitica strains, in which bacilli bearing the virulence plasmid predominate, exhibit all tested phenotypic properties whereas the plasmid-cured isogenic strains show no one of these features. Therefore all the tested markers could be useful for detection of virulent Y. enterocolitica strains directly isolated from patients. The most useful virulence markers in bacteriological study seems to be calcium dependency and Congo-red binding, examined together by the use of CRMOX agar, because they confirm the presence of the virulence plasmid by parallel detection of two physiologically different features associated with this plasmid. In addition CRMOX agar allows for the examination rough strains while agglutination tests do not.     

Virulence plasmid (pYV)-associated susceptibility of Yersinia enterocolitica to chlorine and heavy metals

This study was aimed at elucidating the role of virulence plasmid (pYV) in the susceptibility of Yersinia enterocolitica to bactericidal agents such as chlorine and heavy metals. Plasmid-bearing (pYV+) Y. enterocolitica was less susceptible to the antimicrobial action of chlorine and heavy metals compared with the isogenic plasmidless (pYV-) derivative. This difference was, however, observed only with bacteria cultured at 25 degrees C. pYV-associated susceptibility apart, cells cultured at 37 degrees C were also found to be less susceptible to the antimicrobial action of these agents. The results indicate that the susceptibility of Y. enterocolitica to these agents was influenced both by the presence of the virulence plasmid and the temperature at which the cells were cultured.     

The Yersinia enterocolitica pYV Virulence Plasmid Contains Multiple Intrinsic DNA Bends Which Melt at 37°C

Temperature has a pleiotropic effect on Yersinia enterocolitica gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (相似文献   

Y. enterocolitica inhibits antigen degradation in dendritic cells

Yersinia enterocolitica (Ye) disrupts the ability of dendritic cells (DC) to prime CD4+ T cells suggesting that Ye may subvert uptake and/or processing of soluble antigens (Ag). To investigate this Ye-infected DC were loaded with fluorescently labelled ovalbumins as markers for Ag uptake and processing, and analysed by flow cytometry, fluorometry and microscopy. Wild type pYV+ as well as plasmidless pYV(-) bacteria inhibited Ag degradation in DC by 40% compared to non-infected cells. Microscopic analyses of pYV(-)-infected DC revealed that 40% of DC contained intracellular bacteria, and that DC without intracellular bacteria had degraded more Ag. When internalization of pYV(-) was blocked by cytochalasin D, Ag degradation was no longer inhibited indicating the competition between degradation of bacteria and ovalbumin. In contrast, cytochalasin D pre-treated DC infected with pYV+ inhibited Ag degradation by a mechanism dependent on the presence of virulence plasmid pYV encoding YopE, YopH, YopM, YopP, YopT and YopO. As no single Yop inhibited Ag degradation, interaction of multiple Yops might account for this effect, possibly by inhibiting Rho GTPases, because of a significant decrease of Ag degradation observed in DC incubated with toxin B of C. difficile. However, the contribution of other pYV-encoded factors cannot be excluded.     

Susceptibility to selected antibiotics of Yersinia enterocolitica 03 strains, carrying and not carrying plasmid pYV

A total of 199 clinical strains of Yersinia enterocolitica serotype O3, biotype 4 were tested for their susceptibility to antibiotics (158 strains carried the virulence plasmid pYV and 41 strains did not). 114 isolates were tested by standard disk diffusion method for 21 antibiotics. Almost all tested strains were resistant to ampicillin and cefazolin and susceptible to amoxycillin/clavulanate, cefaclor, cefamandole, cefuroxime, cefotaxime, ceftriaxone, aztreonam, imipenem, gentamicin, amikacin, netilmicin, tetracycline, doxycycline, chloramphenicol, ciprofloxacin, sulphamethoxazole, co-trimoxazole, trimethoprim and furazolidone. In addition minimal inhibitory concentrations (MICs) of 15 antibiotics were determined by agar dilution method for all 199 strains (158 plasmid positive and 41 strains plasmid negative). Third-generation cephalosporins such as cefotaxime and ceftriaxone and a fluoroquinolone (ciprofloxacin) were the most active antimicrobial agents, tested followed by aztreonam, imipenem, trimethoprim, tetracycline, gentamicin, chloramphenicol, amoxycillin/clavulanate, cefaclor, cefuroxime, amikacin, furazolidone and sulphamethoxazole. The present study demonstrated a high susceptibility of clinical strains of Y. enterocolitica to most of the tested antibiotics. In general, there was no significant difference between susceptibility of virulence plasmid pYV positive and virulence plasmid negative strains to antibacterial agents.     

Pathogenicity of Yersinia enterocolitica biotype 1A

Yersinia enterocolitica strains of biotype 1A lack the known virulence determinants of strains in other categories, including the Yersinia virulence plasmid (pYV), and several chromosomal markers of pathogenicity. For this reason, and also because Y. enterocolitica strains of biotype 1A are frequently isolated from the environment or asymptomatic individuals, these bacteria are often assumed to be avirulent. On the other hand, there is a considerable body of clinical, epidemiological and experimental evidence to indicate that at least some strains of Y. enterocolitica biotype 1A are able to cause gastrointestinal symptoms which resemble those caused by pYV-bearing strains. The availability of a number of experimental systems, including cell culture and animal models of infection, provides an opportunity to identify and characterise the essential virulence determinants of biotype 1A strains.     

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.     

Note: Inhibition of the growth of Yersinia enterocolitica O:3 by the microflora of porcine caecum and ileum in an in vitro model

The growth of Yersinia enterocolitica O:3 was tested in an in vitro model of the porcine intestine at the physiological temperature of 39°C of growing pigs. The model supported a stable population of Y. enterocolitica at a level 10⁸–10⁹ cells ml^-1. Plasmid profile analysis and the Ca²⁺-dependent proportion of the population suggested that the great majority of the Y. enterocolitica population retained the 70 kb virulence plasmid, pYV, throughout the experimental period of 5 d. The growth of Y. enterocolitica was substantially inhibited by the ileal and the caecal flora compared to the growth of the bacterium alone. Yersinia enterocolitica was not isolated after 3 d of cultivation.     

Involvement of the virulence gene products of Yersinia enterocolitica in the immune response of infected mice

The virulence of Yersinia enterocolitica is known to be highly dependent on its virulence plasmid. However, it remains unclear whether the virulence plasmid is engaged also in the induction of cell-mediated immunity that is essential for protective immunity in the host. In this study, we have compared the induction of type 1 helper T cell immunity against Y. enterocolitica using a virulent strain (P+) harboring the pYV plasmid and an avirulent strain (P-) harboring no pYV. Spleen cells from both groups of mice immunized with 1/10 LD50 of P+ strain and those with 1/10 LD50 of P- strain produced a high level of gamma interferon (IFN-gamma) upon stimulation with heat-killed bacteria, and CD4+ T cells were exclusively responsible for IFN-gamma production. When crude Yersinia outer proteins (Yops) were used for antigenic stimulation, IFN-gamma response of immune spleen cells against crude Yops was observed only in mice immunized with P+ strain. Flowcytometric analysis revealed a significant level of increase in IFN-gamma-producing CD8+ T cells as well as the increase in IFN-gamma-producing CD4+ T cells against crude Yops. These results suggest that the virulence plasmid of Y. enterocolitica is involved in the induction of Th1-type of possibly protective T cells in infected mice.     

Unusual, virulence plasmid-dependent growth behavior of Yersinia enterocolitica in three-dimensional collagen gels

As a first approach to establishing a three-dimensional culture infection model, we studied the growth behavior of the extracellular pathogen Yersinia enterocolitica in three-dimensional collagen gels (3D-CoG). Surprisingly, we observed that plasmidless Y. enterocolitica was motile in the 3D-CoG in contrast to its growth in traditional motility agar at 37 degrees C. Motility at 37 degrees C was abrogated in the presence of the virulence plasmid pYV or the exclusive expression of the pYV-located Yersinia adhesion gene yadA. YadA-producing yersiniae formed densely packed (dp) microcolonies, whereas pYVDelta yadA-carrying yersiniae formed loosely packed microcolonies at 37 degrees C in 3D-CoG. Furthermore, we demonstrated that the packing density of the microcolonies was dependent on the head domain of YadA. Moreover, dp microcolony formation did not depend on the capacity of YadA to bind to collagen fibers, as demonstrated by the use of yersiniae producing collagen nonbinding YadA. By using a yopE-gfp reporter, we demonstrated Ca(2+)-dependent expression of this pYV-localized virulence gene by yersiniae in 3D-CoG. In conclusion, this study revealed unique plasmid-dependent growth behavior of yersiniae in a three-dimensional matrix environment that resembles the behavior of yersiniae (e.g., formation of microcolonies) in infected mouse tissue. Thus, this 3D-CoG model may be a first step to a more complex level of in vitro infection models that mimic living tissue, enabling us to study the dynamics of pathogen-host cell interactions.     

In vitro and in vivo expression studies of yopE from Yersinia enterocolitica using the gfp reporter gene

The Yersinia outer protein YopE belongs to the translocated effector proteins of pathogenic yersiniae. We constructed various truncated yopE genes fused to gfp (encoding the green fluorescent protein) to study yopE gene expression and YopE-GFP translocation of Y. enterocolitica in cell culture and mouse infection models. The hybrid gene fusions were co-expressed in Y. enterocolitica (i) on a low-copy plasmid in the presence of the virulence plasmid pYV08 (in trans configuration) and (ii) after co-integration by homologous recombination of a yopE-gfp-carrying suicide plasmid into pYV08 (co-integrate configuration). After 30min of infection of HEp-2 cell monolayers, extracellularly located yersiniae began to emit green fluorescence after excitation. In contrast, internalized bacteria were weakly fluorescent. Translocation of YopE-GFP into HEp-2 cells by attached yersiniae was visualized by optical sectioning of fluorescent HEp-2 cells using confocal laser scanning microscopy and was confirmed by immunoprecipitation of cytosolic YopE-GFP from selectively solubilized HEp-2 cells. The co-translocation of other Yops was not significantly impaired by YopE-GFP as shown by YopH/YopE-mediated suppression of the oxidative burst of infected neutrophils. The time course of yopE-gfp expression (in trans as well as in the co-integrate configuration) in the HEp-2 cell infection model as well as after in vitro induction was studied using a highly sensitive CCD camera and a flow cytometer. Similar results were obtained with a YopE-LUC (firefly luciferase) protein fusion as reporter. After intraperitoneal, intravenous and orogastrical infection of Balb/c mice with the recombinant yersiniae strains, green fluorescing bacteria could be visualized microscopically in the peritoneum, the spleen, the liver and in the Peyer's patches. However, only weakly fluorescent yersiniae were observed in the intestinal lumen. These results were quantified by flow cytometric measurements. The application of gfp as a reporter gene turned out to be promising for the study of protein translocation by protein type III secretion systems and differential virulence gene expression in vivo.     

The presence of the ail gene in clinical strains of Yersinia enterocolitica isolated from stools in Poland and characteristics of gene variant

The non-American variant of ail gene was found independently of the virulence-associated plasmid pYV in all 152 tested clinical Y. enterocolitica O:3 strains isolated from humans in Poland. Data obtained by sequencing exhibited high genetic stability of the ail gene present in the tested isolates obtained from different regions of the country. The PCR-RFLP technique was sufficient for fast identification and characterization of the ail gene variant present in the tested strains.