The effect of the trophic and temperature conditions of cultivation on lipid synthesis by Yersinia pseudotuberculosis

The comparative study of the synthesis lipids in Y. pseudotuberculosis, depending on the conditions of their cultivation (at different temperatures in mineral media and in media, containing organic compounds), has been carried out. As demonstrated in this study, temperature in the main inducing factor, affecting the synthesis of lipids of definite classes and fatty acids, incorporated into these lipids. During the cultivation of Y. pseudotuberculosis in mineral and organic media under the conditions of low temperature their lipid composition remains unchanged, but at 6 degrees C the synthesis of unsaturated fatty acids prevails, while at 37 degrees C saturated fatty acids are mainly synthesized. On mineral media at 37 degrees C bacteria synthesize mostly nonpolar lipids in the form of reserve substances, represented by triglycerides and free fatty acids.     

Study of a lipopolysaccharide from Yersinia pseudotuberculosis of the IVA serotype

The comparative studied on lipopolysaccharides from Yersinia pseudotuberculosis IVA serovar, strains 32 and 31D, have been conducted. The identity of the lipopolysaccharides isolated from these strains has been shown. The structural pattern of the repeating unit of the O-specific side chain of the lipopolysaccharide has been suggested: (Formula: see text)     

The structure of the O-specific polysaccharide chain of the lipopolysaccharide of Yersinia pseudotuberculosis serovar VII

An O-specific polysaccharide of Yersinia pseudotuberculosis serovar VII has been isolated and characterized. The polysaccharide consists of colitose, D-glucose and 2-acetamido-2-deoxy-D-galactose in the ratio 1 : 2 : 2. From the results of methylation analysis, partial acid hydrolysis, 1H and 13C NMR spectroscopy the structure of the repeating unit of the O-specific polysaccharide is deduced as follows:     

Structure of O-specific polysaccharide from Yersinia pseudotuberculosis of serotype VI lipopolysaccharide

Using methylation studies, partial hydrolysis and 13C NMR spectroscopy data, the following structure of O-specific polysaccharide from lipopolysaccharide of Yersinia pseudotuberculosis VI serovar has been proposed: (Formula: see text).     

The biosynthesis and biological role of 6-deoxyheptose in the lipopolysaccharide O-antigen of Yersinia pseudotuberculosis

Yersinia pseudotuberculosis O:2a harbours 6-deoxy- d - manno -heptose in its O-antigen. The biological function of 6-deoxyheptose and its role in virulence is unknown and its biosynthetic pathway has not been demonstrated experimentally. Here, we show that dmhA and dmhB are necessary for 6-deoxyheptose biosynthesis in Y. pseudotuberculosis . Their disruption resulted in the lack of 6-deoxyheptose in the O-unit and its replacement by d - glycero - d - manno -heptose, thus indicating relaxed specificity of the glycosyltransferases, polymerase and ligase involved in lipopolysaccharide synthesis. The dmhB mutant exhibited a lower content in ketooctonic acid (Ko)-containing core molecules and reduced ligation and polymerization of the O-unit. We also show that Tyr128 is essential for activity of DmhB, and that DmhB functions as an oligomer, based on the dominant negative effect of overexpression of DmhB Y128F in dmhA . Moreover, we demonstrate that 6-deoxyheptose is important for virulence-related functions of the outer membrane and its appendages in vitro , such as barrier function against bile salts, polymyxin and novobiocin, and flagella-mediated motility. Although both mutants colonized the mouse ceacum as well as the wild type, the dmhB mutant was impaired for colonization of the liver, suggesting that DmhB represents a potential therapeutic target.     

Structural studies on the immunodominant group of lipid A from lipopolysaccharide of Yersinia pseudotuberculosis.

Lipid A isolated from lipopolysaccharide of Yersinia pseudotuberculosis was used for immunization of rabbits to afford antisera to lipid A with titers of 1:640 in the passive hemolysis test. Exhaustion of immune serume with sheep erythrocytes decreased antibody titers up to 1:160. Authentic samples of 2-(DL-3-hydroxytetradecanoyl)amino-2-deoxy-D-glucose 6-phosphate, 2-tetradecanoylamino-2-deoxy-D-glucose 6-phosphate and 2-acetamido-2-deoxy-D-glucose 6-phosphate have been synthesized in order to carry out a comparative study of inhibitory activity of these compounds and lipid A using a system of lipid A and antiserum to lipid A. As a result, the immunodominant moiety of the lipid A of Y. pseudotuberculosis proved to contain a D-glucosamine residue acylated with 3-hydroxytetradecanoic acid at the amino group. The nature of the fatty acid acylating the amino group of glucosamine does not play an important role in the structure of immunodominant moiety of lipid A.     

Structure of O-specific polysaccharide isolated from the Yersinia pseudotuberculosis serotype 1A lipopolysaccharide

An O-specific polysaccharide from the lipopolysaccharide Yersinia pseudotuberculosis 1A serovar has been isolated and characterized. This compound was shown to contain residues of paratose, 6-deoxy-D-manno-heptose, D-galactose and 2-amino-2-deoxy-D-glucose in equimolar ratios. Using methylation studies, partial acid hydrolysis and 13C NMR spectroscopy, the following structure was proposed for the repeating unit of the O-specific polysaccharide: (Formula: see text).     

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.     

Electron microscopic study of the interaction of Yersinia pseudotuberculosis with macrophages and HeLa cells

The comparative electron-microscopic study of early stages of the interaction of Y. pseudotuberculosis virulent strain (No. 282) with "professional" (macrophages) and "nonprofessional" (HeLa cells) phagocytes has been carried out. The character of the intimate mechanism of this interaction has been found to be essentially different. The common feature for both systems is the adsorption of bacteria and their penetration into cells due to phagocytosis. But the subsequent fate of Y. pseudotuberculosis is different. In HeLa cells they are isolated from the cytoplasm by multilayer membrane structures, thus remaining morphologically intact. In macrophages the destruction of the microbe in phagolysosomes occurs.     

Differentiation of Yersinia pestis and Y. pseudotuberculosis by SDS-PAGE analysis of lipopolysaccharide

A method is described for the differentiation of Yersinia pestis from Y. pseudotuberculosis , based on the examination of lipopolysaccharide by SDS-PAGE and silver staining.     

13C-NMR spectrum of O-specific polysaccharide from the lipopolysaccharide of Yersinia pseudotuberculosis of serotype III

A 13C NMR spectrum of O-specific polysaccharide isolated from Yersinia pseudotuberculosis III serovar lipopolysaccharide has been interpreted. This allowed to define more precisely the configuration of glycosidic bonds and to confirm the structure of the repeating unit of the specific polysaccharide which was earlier established by other methods.     

Plasmids of Yersinia pseudotuberculosis

Plasmids with the sizes of 5.7; 51; 70-77; and 120-130 kb were found in six strains among the ten strains collection of Yersinia pseudotuberculosis. The restriction endonucleases analysis. Southern-blot hybridization and physical maps construction were performed for the plasmids. The 70-77 kb plasmids were found to be analogous to the Ca2(+)-dependence plasmid pYVO19 from Yersinia pestis EV76. The difference between the plasmids of this type is in the insertions or deletions located on the similar fragments of the restriction maps. The 51 kb plasmid has no common fragments with the Ca2(+)-dependence plasmids and does not code for virulence properties of the strain harbouring it. No homology is shared by the 5.7 kb plasmid and the 10 kb plasmid from Yersinia pestis EV76. Replicon of the 5.7 kb plasmid has been used to construct the pVS11 vector plasmid.     

Structure of the O-specific polysaccharide chain of the Yersinia pseudotuberculosis lipopolysaccharide (serovar II C)]

An O-specific polysaccharide has been isolated on mild acid hydrolysis of lipopolysaccharide from Yersinia pseudotuberculosis serovar IIc and shown to consist of abequose, D-mannose and 2-acetamido-2-deoxy-D-galactose residues in the ratio 0.8:3:1. From the results of acid hydrolysis, 13C NMR, methylation and periodate oxidation studies the structure of the repeating unit of the O-specific polysaccharide is deduced as follows: (formula; see text)     

Effect of acidin-pepsin on Yersinia pseudotuberculosis

The effect of acidin-pepsin solution at a concentration of 1 : 100 on newly isolated Yersinia cultures has been tested in three series of experiments. Acidin-pepsin has been found to exert a bactericidal effect on Yersinia and to induce the appearance of involutionary forms and large rod-shaped Yersinia cells with a better capacity for survival. The surviving Yersinia cells do not pass their resistance to acidin-pepsin on to their progeny and show low invasiveness and pathogenicity in white mice.     

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.     

The biological action of Yersinia pseudotuberculosis endotoxin

Experimental studies on guinea pigs have shown that Y. pseudotuberculosis lipopolysaccharide is capable of inducing endotoxinemia accompanied by the development of the thrombohemorrhagic syndrome. In cases of pseudotuberculosis the importance of the increased synthesis of prostaglandins and cyclic nucleotides with the prevalence of PGF2 alpha and cGMP in the genesis of toxico-allergic manifestations of pathologic processes has been established. The pathomorphological picture of pseudotuberculosis endotoxinemia is characterized by sludge, the vascular thrombosis of the microcirculatory bed, diapedetic hemorrhages, delymphatization of the immunogenetic organs. The moderately pronounced action of indomethacin, a prostaglandin inhibitor, on the manifestations of pseudotuberculosis endotoxinemia has been revealed.     

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.