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A method has been developed for the quantitative estimation of the binding force of a model microsphere with a eukaryocyte based on the optical trap in order to study the molecular mechanism of adhesion between an individual bacterium and a host cell. The substantial role of LPS O-side chains in the adhesiveness of Yersinia pseudotuberculosis 1b to J774 macrophages has been revealed with the use of a set of microspheres functionalized with lipopolysaccharide (LPS) preparations and antibodies with different specificities. The results indicate the significance of the O-antigen as a pathogenicity factor of Y. pseudotuberculosis in colonization of a macroorganism. The developed methodical approaches can be applied to the study of molecular mechanisms of the pathogenesis of pseudotuberculosis and other infectious diseases to improve antiepidemic service.

Aim: Evaluate immune response in mice against various L-asparaginases and determine their cross-immunogenicity., Materials and Methods: The studies were carried out in C57Bl(6j) line mice. Immunogenicity of L-asparaginases was studied: Escherichia coli type II (recombinant) (Medak, Germany) (EcA); Erwinia carotovora type II (ErA); Yersinia pseudotuberculosis type II (YpA); Rhodospirillum rubrum type I (RrA); Wollinella succinogenes type II (WsA). Immune response against the administered antigens was determined in EIA., Results: Y. pseudotuberculosis L-asparaginase was the most immunogenic, E. coli--the least immunogenic. E. carotovora, R. rubrum, W. succinogenes asparaginases displayed intermediate immunogenicity. The results of cross-immunogenicity evaluation have established, that blood sera of mice, that had received YpA, showed cross-immunogenicity against all the other L-asparaginase preparations except E. carotovora. During immunization with E. coli L-asparaginase the developed antibodies also bound preparation from E. carotovora. Sera from mice immunized with W. succinogenes, E. carotovora and R. rubrum L-asparaginases had cross-reaction only with EcA and did not react with other preparations., Conclusion: Cross-immunogenicity of the studied L-asparaginases was determined. A sequence of administration of the studied preparation is proposed that allows to minimize L-asparaginase neutralization by cross-reacting antibodies.

A study of the influence of exogenous factors on the immunochemical activity of the bacterium Yersinia pseudotuberculosis and lipopolysaccharide preparations isolated from bacteria was performed using monoclonal antibodies. It was shown that the hybridomas that were obtained in this work produce antibodies against different and, most likely, species-specific epitopes associated with lipopolysaccharide O side chains. The antibody concentrations produced increased with a decrease in the temperature, at which the bacteria were cultivated. An inhibitory effect of proteinase K, pepsin, and trypsin on the immunochemical activity of bacterial cells, determined using a solid-phase enzyme immunoassay, was demonstrated. Treatment with sodium periodate showed no uniform effect on the reactions between monoclonal antibodies and antigens (lipopolysaccharides and microbial cells), as adjudged by an immunoassay, which is most likely a consequence of the different localization of lipopolysaccharide epitopes recognized by the antibodies from four hybridomas.

An enzyme immunoassay-based test system for Y. pestis V antigen detection was developed. The specificity and sensitivity of this system met the requirements for medical immunobiological preparations for the identification of causative agents of highly fatal diseases. The sensitivity of the test system was assessed, and its high specificity was also demonstrated: the test system did not detect bacterial cells of closely related (four Y. pseudotuberculosis strains) and heterologous microorganism strains. The test system developed was able to detect the V antigen at concentrations as low as 2.0 ng/mL in cells of nine experimental Y. pestis cultures. The obtained preparation can be recommended for use in laboratory diagnostics of plaque.

Aim: Evaluation of immunogenic and protective properties of constructs based on subunit porin antigen from Yersinia pseudotuberculosis, immunostimulating complexes (ISCOM) and tubular immunostimulating (TI) complexes., Materials and Methods: Porin antibodies and blood serum cytokines were determined by using EIA. Porin-specific cell immunity was evaluated by DTH reaction inflammation index. Protective activity of porin formulations was determined by measuring specific gravity of animals surviving Yersinia pseudotuberculosis lethal challenge., Results: Porin in TI complexes develops higher immunogenicity when compared with individual protein or protein with complete Freunds adjuvant. Porin in TI complexes develops higher protective activity, inhibits interferon synthesis in mice. Incorporation of porin into TI complexes results in neutralization of porin suppressive activity against DTH mechanisms and interferon system., Conclusion: TI complexes may be used as perspective carriers for bacterial antigens. TI complexes have adjuvant properties and can provide protective properties to porin vaccine constructs.

When cultivated in the presence of glucose, irrespective of temperature and the degree of aeration, Y. pseudotuberculosis cells have the ovoid form, constant size and low hydrophobic properties of their surface. Meanwhile the characteristics of the bacteria grown in the medium, carbohydrate-free or with galactose added, essentially depend on the conditions of medium aeration. Under the conditions of intensive stirring at both temperatures these bacteria acquire the coccoid form, not typical for Yersinia, they have a smaller area (approximately 2 times) and more hydrophobic surface in comparison with the cells grown in the presence of glucose. Under stationary conditions the differences between the cells, cultivated in the presence of galactose and glucose, in form and area disappear, but the differences in the hydrophobic properties of the surface are retained. As revealed in this study, the cells grown in the presence of galactose and under the conditions of intensive medium stirring, in contrast to those grown with glucose, have 1.5-fold greater invasive activity, irrespective of aeration conditions, eightfold greater resistance to ampicillin and twofold greater resistance to streptomycin and erythromycin.

Certain physicochemical properties of glycoglycerolipids from marine alga Laminaria japonica (monogalactosyl diacylglycerol, digalactosyl diacylglycerol, and sulfoquinovosyl diacylglycerol) and their ability to be incorporated into immunostimulating complexes (ISCOMs) used for presentation of microbial and tumor antigens in vesicular form were comparatively described. These glycolipids proved to considerably differ by fatty acid composition, degree of unsaturation, and phase transition temperature. Possible production of modified ISCOMs through incorporation of these glycolipids into the vesicle instead of the glycolipid component was demonstrated. Preliminary data demonstrated no significant increase in immune response to Yersinia pseudotuberculosis porin within the modified (with monogalactosyl diacylglycerol) and classical (with phosphatidylcholine) ISCOMs as compared to individual porin.

The effects of the culturing method (suspension cultures in a liquid nutrient broth or colonies on a solid agarized medium) and the growth phase on the lypopolysaccharide (LPS) composition of Yersinia pseudotuberculosis (O:Ib serovar, strain KS 3058) grown in cold (5 degrees C) were studied. The amount the LPS synthesized by cells depended on the bacteria growth phase for both media. The LPS acylation degree was constant, whereas the length of the O-specific polysaccharide chain varied with the culture age and achieved maximum in the stationary growth phase for both media. The bacteria culturing on the nutrient agar stimulated more intensive synthesis of LPS, which were extracted more easily, had longer polysaccharide O-chains, and were more toxic than LPS of the bacteria cultured in the liquid medium. It was proposed that the culturing of Yersinia pseudotuberculosis in cold as colonies on the agar surface causes an increase in the bacterial virulence.

The ability to correct activity of Y. pseudotuberculosis thermoresistant toxin on antioxidative enzymes and on active oxygen forms in neurophiles and mononuclears was investigated. Toxin at concentration 0.5 and 2.5 mcg/ml did not change O2- production and activity of superoxidedismutase (SOD) and glutathione peroxidase (GP), but significantly enhanced catalase and glutathiont reductase (GR) activity. Gangleen at concentration 0.0002-0.2 mcg/ml, when added to incubation medium with toxin, stimulated production of active oxygen forms and activity of SOD, catalase, GP in both types of leucocytes, but decreased activity of GR in mononuclears. The results of investigation proves the ability to use gangleen in correction of immune system disorders caused by Y. pseudotuberculosis thermoresistant toxin.

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.

The possibility of preparing the lipid A (LA) from Yersinia pseudotuberculosis Serovar IB by the hydrolysis of whole cells instead of the preliminary isolation of lipopolysaccharide (LPS) was demonstrated. Direct extraction with an organic solvent of the bacterial mass preliminary treated with 10% acetic acid or 1 M HCl was shown to result in a di- (LAAcOH) or monophosphoryl derivative (LAHCl), respectively. These were completely extractable only after treatment with strong hydrolyzing agents. We concluded that two forms of LA (and LPS) exist in the pseudotuberculosis bacterium which differ in the stability of their bonding to the bacterial outer membrane.

Changes in the structure and functional activity of porin, a protein from Yersinia pseudotuberculosis, resulting from the removal of lipopolysaccharide (LPS) normally bound with the protein were studied. The treatment of LPS-containing porin with a 30% SDS solution led to an LPS-free protein that, according to the SDS-PAGE, remained to be a trimer. It was shown by CD and UV spectroscopies and intrinsic protein fluorescence that the removal of LPS caused only conformational changes in the porin secondary and tertiary structures. The LPS-free porin folded into a completely beta-structured protein aggregate. The bilayer lipid membrane technique showed that the pore-forming activity of the LPS-free porin decreased, and its concentration should be increased by two orders of magnitude to achieve the same effect. Incubation of the LPS-free porin with LPS led to a porin-LPS complex and affected the character of the protein functional activity. The treatment of the LPS-free porin by octyl glucoside, a nonionic detergent, resulted in the restoration of the protein pore-forming activity. It was suggested that the LPS and detergent provide a definite protein conformation necessary for its functioning.

For the first time the metabolic processes, taking place during "cold" cultivation of Y. pseudotuberculosis and involving organic and amino acids, were shown to be more economical, thus permitting the bacteria to maintain its viability for a rather long time at low temperature without the need to consume these substances from outside. At higher temperature the bacteria switches its metabolic paths to the consumption of nutrient substances in greater amounts from outside, increasing its constructive metabolism.

The interaction of the pore-forming protein (porin) from the outer membrane of Yersinia pseudotuberculosis with the S- and R-forms of lipopolysaccharide (LPS) from this bacterium was studied. Analysis of the equilibrium binding of 125I-labeled S- and R-LPS, as well as the competitive inhibition of this reaction by lipid A, core oligosaccharide, and O-specific polysaccharide, suggests that there are binding sites on the porin molecule specific to these fragments of the LPS molecule. The binding of R-LPS occurs at independent sites of two classes with Ka 1.7 x 10(5) and 1.1 x 10(5) M-1. S-LPS interacts with porin with positive cooperation (h 1.6) and Ka 0.8 x 10(5) M-1. The number of binding sites was found to be nine and four R- and S-LPS, respectively. Molecular mechanisms of the interaction are discussed.

By means of physico-chemical methods, lipid bilayer reconstitution and immunoenzyme assay, macromolecular organization of porin oligomers from Yersinia pseudotuberculosis, isolated by two extraction methods, was studied. Use of SDS and high temperature in the course of the extraction led to a partial denaturation of porin trimers at the level of the tertiary structure, these conformational changes affecting the porin's pore-forming activity and antigenic structure. At the same time, the partially denatured trimers are as stable under the treatment of urea and guanidine hydrochloride as the native protein.

Y. pseudotuberculosis strains were grown at 6 degrees-8 degrees C and then incubated at 37 degrees C. 3-6 hours later serum resistance appeared in the strains having plasmid virulence and producing outer membrane polypeptide with a molecular weight of 120 kD, known as P1. 10-12 hours later serum resistance appeared in the strain having the virulence plasmid, but not producing P1, as well as in strains in which the plasmid was eliminated.