[INTESTINAL FAILURE AND YERSINIA PSEUDOTUBERCULOSIS TRANSLOCATION IN THE DEVELOPMENTOF EXPERIMENTAL GENERALIZED INFECTION].

THE OBJECTIVE: To determine the value of intestinal failure and translocation of bacteria Y. pseudotuberculosis, and normal intestinal microbiota in the initiation and generalization of infection in experimental pseudotuberculosis in conventional white mice, as well as pathological manifestation of it as a response to the adhesion and colonization of the mucosus membrane by pathogenic bacteria Y. pseudotuberculosis. MATERIALS AND METHODS: Experimental models of pseudotuberculosis in conventional white mice used the pathogenic Y. pseudotuberculosis 147 serotype I strain, containing a calcium-dependence plasmid with a molecular weight of 47 MDa. Cultivation of the pseudotuberculosis pathogen given its psychrophilic was performed on Hottinger agar at a temperature of (4-5) °C. The lactobacilli strain L plantarum 8P-A3 was isolated from a lyophilized commercial probiotic Lactobacterin (manufactured by "NPO Microgen", Russia) and used to obtain native culture supernatant fluid of lactobacilli, the composition of which was detected by gas-liquid chromatography with mass-selective detection. Gentamicin for parenteral administration was manufactured by JSC "Biochemist", Russia. Pathomorphological examination was performed on the 4-6th day of the experiment. Fragments of the small intestine, liver, kidneys, and lungs from dead animals were chosen for examination. Tissues were fixed in 10% neutral formalin, dehydrated in isopropanol and embedded in paraffin. Preparations were stained with Ehrlich hematoxylin and eosin, examined on the microscope "Mikmed-2" (JSC "LOMO", Russia) under magnification x 200-x1000. Statistical processing of the experimental results was carried out according to the method of Kerber in modification of I.P. Ashmarin and A.A. Vorobyov. RESULTS: The role of intestinal failure and translocation of bacteria Y. pseudotuberculosis, and normal intestinal microbiota in the initiation and generalization of infection in animals has been found. It has been proved that the oral administration of supernatant fluid containing microbial metabolites to animals as intramuscular administration of gentamicin equally prevent the development of generalized pseudotuberculosis and intensity of pathomorphological changes in the intestine and other organs of animals. CONCLUSION: Metabolites of the probiotic lactobacilli strain L plantarum 8P-A3 jugulate the development of pseudotuberculosis at an early stage of the pathological process in experimental animals infected with pathogen Y pseudotuberculosis, not only causing the preservation of the colonization resistance of the intestinal mucosa that prevents the adhesion and colonization of the pathogen, but also through their antimicrobial impact on the dissiminated pseudotuberculosis patho-gen bacteria in animals.

Alternative endogenous protein processing via an autophagy-dependent pathway compensates for Yersinia-mediated inhibition of endosomal major histocompatibility complex class II antigen presentation.

Extracellular Yersinia pseudotuberculosis employs a type III secretion system (T3SS) for translocating virulence factors (Yersinia outer proteins [Yops]) directly into the cytosol of eukaryotic cells. Recently, we used YopE as a carrier molecule for T3SS-dependent secretion and translocation of listeriolysin O (LLO) from Listeria monocytogenes. We demonstrated that translocation of chimeric YopE/LLO into the cytosol of macrophages by Yersinia results in the induction of a codominant antigen-specific CD4 and CD8 T-cell response in orally immunized mice. In this study, we addressed the requirements for processing and major histocompatibility complex (MHC) class II presentation of chimeric YopE proteins translocated into the cytosol of macrophages by the Yersinia T3SS. Our data demonstrate the ability of Yersinia to counteract exogenous MHC class II antigen presentation of secreted hybrid YopE by the action of wild-type YopE and YopH. In the absence of exogenous MHC class II antigen presentation, an alternative pathway was identified for YopE fusion proteins originating in the cytosol. This endogenous antigen-processing pathway was sensitive to inhibitors of phagolysosomal acidification and macroautophagy, but it did not require the function either of the proteasome or of transporters associated with antigen processing. Thus, by an autophagy-dependent mechanism, macrophages are able to compensate for the YopE/YopH-mediated inhibition of the endosomal MHC class II antigen presentation pathway for exogenous antigens. This is the first report demonstrating that autophagy might enable the host to mount an MHC class II-restricted CD4 T-cell response against translocated bacterial virulence factors. We provide critical new insights into the interaction between the mammalian immune system and a human pathogen.

[Clinical and prognostic criteria for different forms and types of the course of Yersinia infection].

AIM: to reveal the prognostically significant symptom complexes of pseudotuberculosis/yersiniasis in their different forms and types. SUBJECTS AND METHODS: A comprehensive examination and a long-term follow-up were made in 295 patients with yersiniasis and pseudotuberculosis. A special score scale was developed to evaluate the prognostic value of their clinical symptoms. RESULTS: The symptom complexes prognostically significant to the outcome of Yersinia infection were found. CONCLUSION: The use of the described prognostically significant symptom complexes of Yersinia infection makes it possible to correctly evaluate the course of the disease in each case, identify risk-group patients, and to use adequate treatment.

Regulation of Yersinia Yop-effector delivery by translocated YopE.

The bacterial pathogen Yersinia pseudotuberculosis uses a type III secretion (T3S) system to translocate Yop effectors into eukaryotic cells. Effectors are thought to gain access to the cytosol via pores formed in the host cell plasma membrane. Translocated YopE can modulate this pore formation through its GTPase-activating protein (GAP) activity. In this study, we analysed the role of translocated YopE and all the other known Yop effectors in the regulation of effector translocation. Elevated levels of Yop effector translocation into HeLa cells occurred by YopE-defective strains, but not those defective for other Yop effectors. Only Yersinia devoid of YopK exhibits a similar hyper-translocation phenotype. Since both yopK and yopE mutants also failed to down-regulate Yop synthesis in the presence of eukaryotic cells, these data imply that translocated YopE specifically regulates subsequent effector translocation by Yersinia through at least one mechanism that involves YopK. We suggest that the GAP activity of YopE might be working as an intra-cellular probe measuring the amount of protein translocated by Yersinia during infection. This may be a general feature of T3S-associated GAP proteins, since two homologues from Pseudomonas aeruginosa, exoenzyme S (ExoS) and exoenzyme T (ExoT), can complement the hyper-translocation phenotypes of the yopE GAP mutant.

[Yersinia pseudotuberculosis toxins].

The review of publications about protein toxins Y. pseudotuberculosis are presented. It includes the main data obtained by domestic and foreign investigators as well as the results of our own elaboration in the study of Y. pseudotuberculosis protein toxins. The guestions of isolation, purification, characterization of physico-chemical and biological properties, the mechanism action and role of toxins on pathogenesis of infection were discussed.

[The nature, frequency, specificity and duration of the retention of changes in different parts of the gastrointestinal tract in yersiniosis]. / Kharakter, chastota, spetsifichnost' i dlitel'nost' sokhraneniia izmenenii v razlichnykh otdelakh zheludochno-kishechnogo trakta pri iersiniozakh.

400 patients with yersiniosis, shigellosis, salmonellosis and abdominal typhoid were examined clinically, roentgenologically and using various instrument techniques for changes in the stomach and intestine. Yersiniosis and abdominal typhoid were characterized by dyskinesia of the small intestine and ileotyphlitis, Flexner's shigellosis by catarrhal-hemorrhagic proctosigmoiditis, Sonne salmonellosis and shigellosis by enteritis. Terminal ileitis occurred more frequently in yersiniosis patients in recurrence. After yersiniosis the patients may retain (for 3-24 months) lesions of ileocecal intestine without or rare clinical symptoms and develop chronic disease similar to initial stage of Crohn' disease.

[The functional status and work capacity of convalescents after pseudotuberculosis]. / Funktsional'noe sostoianie i rabotosposobnost' rekonvalestsentov posle psevdotuberkuleza.

The authors have studied the recovery process of functional state and performance capability in 887 convalescents after pseudotuberculosis. All the examinees were servicemen of male sex, age 18-23, without somatic or psychoneurological disorders. Clinical, biochemical, immunological and psychophysiological methods of examinations were applied. It was found out that the outcomes of the diseases were favourable. At the same time in 79.8% of patients functional disorders of nerve, cardiovascular, hepatobiliary and digestive systems were taking place for one month. A half of convalescents had a low performance capability and low psychoemotional labour motivation. Application of adaptogens, physiotherapy, and a system of gradually growing physical loads which was elaborated by the authors give the possibility to improve and shorten the period of rehabilitation.

The proinflammatory response induced by wild-type Yersinia pseudotuberculosis infection inhibits survival of yop mutants in the gastrointestinal tract and Peyer's patches.

Single-strain infections and coinfections are frequently used to assess roles of virulence factors in infected tissues. After oral inoculation of mice, Yersinia pseudotuberculosis yopE and yopH mutants colonize the intestines and Peyer's patches in single-strain infections but fail to persist in competition with wild-type Y. pseudotuberculosis, indicating that these two infection models provide different insights into the roles of Yops. To determine how wild-type Y. pseudotuberculosis hinders yop mutant survival, yop mutant colonization and host responses were investigated in several different infection models that isolated specific features of wild-type Y. pseudotuberculosis infection. Infection with wild-type Y. pseudotuberculosis caused significantly more inflammation than yop mutants. Results from coinfections of gamma interferon (IFN-gamma)-/- mice revealed that IFN-gamma-regulated defenses target these mutants, suggesting that YopE and YopH protect Y. pseudotuberculosis from these defenses in BALB/c mice. We developed an oral-intraperitoneal infection model to evaluate the effects of spleen and liver colonization by Y. pseudotuberculosis on yop mutants in the intestines. Spleen and liver infection increased inflammation and decreased yop mutant survival in the intestines, indicating that infection of these organs has consequences in intestinal tissues. Finally, competition infections with Y. pseudotuberculosis mutants with various abilities to induce inflammation demonstrated that survival of the yopE, but not the yopH, mutant was consistently decreased in inflamed tissues. In summary, infection with Y. pseudotuberculosis in intestinal and systemic sites induces intestinal inflammation, which decreases yop mutant survival. Thus, competition studies with wild-type yersiniae reveal critical roles of Yops in combating host responses to a normal virulent infection.

Comparison of YopE and YopT activities in counteracting host signalling responses to Yersinia pseudotuberculosis infection.

Pathogenic Yersinia species share a type III secretion system that translocates Yop effector proteins into host cells to counteract signalling responses during infection. Two of these effectors, YopE and YopT, downregulate Rho GTPases by different mechanisms. Here, we investigate whether YopT and YopE are functionally redundant by dissecting the contribution of these two effectors to the pathogenesis of Yersinia pseudotuberculosis in a mouse infection and tissue culture model. Four days after oral infection, a YopE(+) T (-) strain and a YopE(+) T (+) strain colonized spleens of mice at similar levels, suggesting that YopT is not required for virulence. In contrast, spleen colonization by a YopE(-)T(-) strain was significantly reduced. A YopE(-) T (+) strain colonized spleen at levels comparable to those of the YopE(+) T (-) strain, arguing that YopT can promote virulence in the absence of YopE. Infection of HeLa cells with a YopE(-) T(-)H(-)J(-) strain expressing either YopE or YopT showed that YopE had a stronger antiphagocytic activity than YopT. Expression of YopE strongly inhibited activation of JNK, ERK and NFkappaB, and prevented production of IL-8; whereas YopT moderately inhibited these responses. On the other hand, pore formation was inhibited equally by YopE or YopT. In conclusion, YopE is a potent inhibitor of infection-induced signalling cascades, and YopT can only partially compensate for the loss of YopE.

A Caenorhabditis elegans model of Yersinia infection: biofilm formation on a biotic surface.

To investigate Yersinia pathogenicity and the evolutionary divergence of the genus, the effect of pathogenic yersiniae on the model organism Caenorhabditis elegans was studied. Three strains of Yersinia pestis, including a strain lacking pMT1, caused blockage and death of C. elegans; one strain, lacking the haemin storage (hms) locus, caused no effect. Similarly, 15 strains of Yersinia enterocolitica caused no effect. Strains of Yersinia pseudotuberculosis showed different levels of pathogenicity. The majority of strains (76 %) caused no discernible effect; 5 % caused a weak infection, 9.5 % an intermediate infection, and 9.5 % a severe infection. There was no consistent relationship between serotype and severity of infection; nor was there any relationship between strains causing infection of C. elegans and those able to form a biofilm on an abiotic surface. Electron microscope and cytochemical examination of infected worms indicated that the infection phenotype is a result of biofilm formation on the head of the worm. Seven transposon mutants of Y. pseudotuberculosis strain YPIII pIB1 were completely or partially attenuated; mutated genes included genes encoding proteins involved in haemin storage and lipopolysaccharide biosynthesis. A screen of 15 defined C. elegans mutants identified four where mutation caused (complete) resistance to infection by Y. pseudotuberculosis YPIII pIB1. These mutants, srf-2, srf-3, srf-5 and the dauer pathway gene daf-1, also exhibit altered binding of lectins to the nematode surface. This suggests that biofilm formation on a biotic surface is an interactive process involving both bacterial and invertebrate control mechanisms.

The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence.

The Yersinia pseudotuberculosis chromosome contains a seven-gene polycistronic unit (the pmrF operon) whose products share extensive homologies with their pmrF counterparts in Salmonella enterica serovar Typhimurium (S. typhimurium), another Gram-negative bacterial enteropathogen. This gene cluster is essential for addition of 4-aminoarabinose to the lipid moiety of LPS, as demonstrated by MALDI-TOF mass spectrometry of lipid A from both wild-type and pmrF-mutated strains. As in S. typhimurium, 4-aminoarabinose substitution of lipid A contributes to in vitro resistance of Y. pseudotuberculosis to the antimicrobial peptide polymyxin B. Whereas pmrF expression in S. typhimurium is mediated by both the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems, it appears to be PmrA-PmrB-independent in Y. pseudotuberculosis, with the response regulator PhoP interacting directly with the pmrF operon promoter region. This result reveals that the ubiquitous PmrA-PmrB regulatory system controls different regulons in distinct bacterial species. In addition, pmrF inactivation in Y. pseudotuberculosis has no effect on bacterial virulence in the mouse, again in contrast to the situation in S. typhimurium. The marked differences in pmrF operon regulation in these two phylogenetically close bacterial species may be related to their dissimilar lifestyles.