Comparative evaluation of mono- and neutrophilokins inducing activity of Yersinia pestis antigens

The results of the comparative analysis of the cytokine inducing activity of Yersinia pestis EV antigens are presented. Y. pestis fraction 1A (F1A) and lipopolysaccharide (LPS) were shown to induce mono- and neutrophilokines, regulating cooperative interaction of phagocytes in the process of immunity formation to plague. Neutrophilokines and monokines exceed in their capacity for inducing F1A such acknowledged inductor as Escherichia coli LPS. As revealed by the comparative evaluation of Y. pestis EV LPS and E. coli LPS, neutrophilokines synthesized under the action of the former preparation, have greater influence on the inhibition of the macrophage migration from the infection focus as well as on digestive activity of these cells (in secondary immune response) and on the labilization of the lysosome membranes of macrophages than neutrophilokines induced by E. coli LPS. At the same time they produce a lesser modulating effect on the killer and chemotactic activity of neutrophils, as well as on the expression of FC receptors (FcR) on their surface in comparison with monokines, synthesized under the influence of E. coli LPS.     

Monokine-producing activity of human monocyte-like cell line U937 induced by Yersinia pestis EV antigens

The data on a study of the monokine-producing ability of human monocyte-like cell line U937 are presented. Antigens of Yersinia pestis EV (lipopolysaccharide and fraction 1A) induce monokine production by cell line U937. The obtained monokines essentially enhance neutrophil killer and chemotactic activities, stimulate FcR expression, increase the number of lysosomes, and the lability of lysosomal membranes in neutrophils. F1A significantly suppresses LPS in respect to the ability to induce monokine production, which stimulate neutrophil functional activity.     

Evaluation of vaccinal immunogenesis by the peroxidase method

The possibility of evaluating functional immunomorphogenesis in the course of the vaccinal process after the injection of conjugated and live brucellosis vaccines, as well as conjugated plague antigen and Yersinia pestis strain EV, to guinea pigs has been shown by means of the direct and two-layer variants of the immunoperoxidase assay. The dynamics of the accumulation of globulin-producing cells in the immunocompetent organs and the time course of immunoglobulin titers in the peripheral blood after the injection of live and conjugated vaccines have been followed up. These data may be used for the morphological evaluation of approved preparations.     

Lectin of Yersinia pestis vaccine strain EV and its immunological properties

As the result of the chromatographic separation of Y. pestis EV membrane proteins, a protein fraction with hemagglutinating activity was obtained. The isolated preparation was glycoprotein with a molecular weight of 22 kD, contained 16% of carbohydrates and exhibited thermolabile properties. The determination of the carbohydrate specificity of this glycoprotein revealed that it belonged to the class of lectins. Changes in the content of 11 corticosteroids and the population composition of lymphocytes, as well as the detection of specific antibodies in the blood serum of guinea pigs immunized with lectin, were indicative of the fact that the preparation was sufficiently immunogenic and induced the activation of the processes of proliferation and activation of lymphocytes during immunogenesis. The lectin isolated from Y. pestis EV outer membrane may be regarded as an additional factor ensuring the contact of the pathogen with the cells of the body and as a promising component of combined plague vaccine.     

Changes in the functional activity of neutrophils during their interaction with Yersinia pestis in vitro

The functional state and electrochemical properties of human blood neutrophil leukocytes after their in vitro interaction with Y. pestis cells, strain EV, was analyzed. A considerable decrease in the electrophoretic mobility of neutrophil leukocytes and a considerable increase in their phagocytic indices was shown. At the same time the maximum phagocytic activity in the total pool of isolated neutrophil leukocytes was registered in fractions having higher electrophoretic mobility. The dependence of electrophoretic mobility on the state of neutrophil membranes, as well as the degree of their activation, is discussed.     

Immunogenicity of Yersinia pestis grown on nutrient media at 28 and 37 degrees C

The immunogenicity of Y. pestis strain EV, grown in yeast-casein medium, yeast medium with Hottinger digest and yeast medium with sunflower-seed protein at 28 degrees C and 37 degrees C, for guinea pigs and white mice has been studied. As revealed in this study, these media ensure the formation of highly immunogenic populations of Y. pestis strain EV and, therefore, can be used for growing Y. pestis vaccine strains. Considerable fluctuations in the content of such highly protective antigen as fraction 1 do not affect the immunogenicity of live cultures of Y. pestis strain EV. This is due to the leveling of differences in the content of this antigen in the process of the multiplication of these bacteria in laboratory animals.     

The properties of the cellular surface of Yersinia pestis strain EV and its achromogenic variants

The comparative study of the properties of the surface of vaccine strain Y. pestis EV and its achromogenic variants (AV) differing from the initial strain by decreased immunogenicity and by the morphology of colonies, has been made. The achromogenicity of Y. pestis colonies has been shown to correlate with the loss of the outer membrane protein with a molecular weight 22 kD. The synthesis of this protein is determined by chromosomal genes. AV have been found to have different sensitivity to bacteriophages. The analysis of the electrokinetic potential of Y. pestis EV and its AV has revealed that in the latter have surface charge is considerably greater (1.4- to 1.5-fold). As shown in this study, the hemagglutinating activity of AV with respect to red blood cells of humans with blood group I (O) and guinea pigs is decreased by 1-2 orders and these strains do not agglutinate with sheep red blood cells. The low activity of the initial stage of the phagocytosis of AV by mouse macrophages has been shown. The possible role of the 22 kD proteins as an adhesion factor is discussed.     

Comparative evaluation of the neutrophilokine-inducing activity of Yersinia pestis EV and Escherichia coli lipopolysaccharides

As shown in this study, neutrophilokine-inducing capacity of Y. pestis EV lipopolysaccharide (LPS) was not inferior to, and in secondary immune response even exceeded, that of E. coli LPS. Neutrophilokines synthesized under the action of the former preparation produced greater influence on the inhibition of macrophage migration from the focus of infection, the phagocytic activity of these cells (in secondary immune response) and the labilization of the lysosomic membranes of macrophages than neutrophilokines induced by E. coli LPS. Only in primary immune response the digestive capacity of macrophages was more actively stimulated by neutrophilokines induced by E. coli LPS. Both preparations did not induce the secretion of neutrophilokines regulating the expression of Fc-receptors on the surface of macrophages.     

Development of a diagnostic test for Yersinia pestis by the polymerase chain reaction

A 501 bp caf1 gene fragment and a 443 bp of pla gene fragment carried by 100 kb (pFra) and 10 kb (pPst) species-specific extrachromosomal replicons, respectively, were used as targets to study the conditions under which DNA amplification by polymerase chain reaction (PCR) may be applied to detect and identify Yersinia pestis DNA in cell lysates of pure cultures and biological samples. The sensitivity limit of PCR with the crude cell lysates of Y. pestis EV was estimated as 10–50 cfu in reaction mixture. When target Y. pestis EV cells were mixed with fresh blood of white mice, which contained 0.4% potassium citrate, the PCR detection level varied from 400 to 100 cfu ml^-1 of blood depending on the method used for preparing the sample. In our tests PCR was effective for the detection of yersinia in the blood of white laboratory mice experimentally infected with virulent Y. pestis KM638 strain. This method can be considered convenient for routine detection and identification of Y. pestis.     

Experimental evaluation of interaction between Yersinia pestis and soil infusoria and possibility of prolonged preservation of bacteria in the protozoan oocysts

The character and outcome of interactions between Y. pestis (vaccine strain and soil infusoria Tetrahymena pyriformis (axenic culture) were under experimental study. The parallel use of the bacteriological method and PCR test systems made it possible to follow the dynamics of Y. pestis cells (strain EV) with different plasmid profiles in their interaction with infusoria, as well as their passage into the protozoa cysts. The study revealed the complete utilization of Y. pestis cells lacking virulence factors by infusoria. The presence of plasmids of virulence influenced only the duration of complete bacterial phagocytosis. A drop in the temperature of cultivation to 2 degrees C induced the mass and rapid encystment of infusoria. In the PCR analysis specific DNA fragments of Y. pestis cells, preserved in the latent (uncultivable) state, were detected in the cysts of protozoa within the period of up to 14 months, while the results of bacteriological studies were negative. The data thus obtained are discussed with regard to the possible mechanisms of the existence and prolonged reservation of Y. pestis in the soils of natural foci with participation of protozoa.     

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28 degrees C and 37 degrees C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Several proteins with molecular weights ranging from 34 kDa to 71 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could also be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discussed.     

Antigenic activity of external membrane proteins in Yersinia pestis EV

The study of immunocomplexes obtained from antisera to the preparations of Y. pestis outer membranes and membrane proteins has revealed that outer membrane proteins are involved in the formation of the immunocomplex and belong to previously unknown Y. pestis EV antigens.     

Mechanism and pathway of penicillopepsin-catalyzed transpeptidation and evidence for noncovalent trapping of amino acid and peptide intermediates

Penicillopepsin acting on Nph-Ala2-amide (where Nph = p-nitrophenylalanyl) catalyzes a transpeptidation reaction which leads to the formation of Nph2-Ala2-amide, which arises from condensation of the substrate with enzyme-bound Nph, as the first product released from the enzyme. This is followed by a stage during which Nph3 and Ala2-amide are the major products. A small amount of Nph4 is also formed during this time. Nph and Nph2, formed during the reactions, are tightly, but probably not covalently, bound to the enzyme. They appear as free products only as a result of the cleavage of Nph3 and Nph4 and after most of the substrate Nph-Ala2-amide has been used up. They act as acceptors for the substrate and for Nph2-Ala2-amide. Nph3-Ala2-amide, formed by condensation of Nph-Ala2-amide or of Nph2-Ala2-amide with enzyme-bound Nph2 or Nph, respectively, is also released but is cleaved rapidly to give Nph3 and Ala2-amide. Incorporation of 18O from [18O]water into the carbonyl oxygens of the products is extensive and shows that release of the intermediates is slower than peptide bond cleavage and peptide bond formation. Hence the rate-limiting step in these reactions is product release. No 18O is incorporated into the initial substrate. We propose that Nph and Nph2 as intermediates are held in the active site by hydrogen bonds and by two strong electrostatic interactions.     

A new purification strategy for fraction 1 capsular antigen and its efficacy against Yersinia pestis virulent strain challenge

F1 antigen is an attractive candidate for the development of a subunit vaccine against plague. In previous study, the extraction of this antigen from Yersinia pestis is characterized by using organic solvents. In this work, a new purification strategy that produced high-purity F1 antigen from Y. pestis EV76 was developed by the substitution of physical disruption for organic solvent one, followed by a combination of ammonium sulfate fractionation and Sephacryl S-200HR column filtration chromatography. As revealed in this study, this purification procedure is simple and effective, and avoids potential adverse effect on the antigen by organic solvents. Highly purified F1 that adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to F1 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10(4) CFU of Y. pestis virulent strain 141.     

微量法鼠疫菌质粒DNA抽提的优化

旨在分析微量法抽提鼠疫菌质粒DNA的效果,探讨其在鼠疫菌分子生物学实验研究中的应用价值.采用微量法分别提取鼠疫菌EV76株,假结核耶尔森菌PstII株及大肠杆菌V517株质粒DNA,琼脂糖凝胶电泳对质粒DNA抽提结果进行分析.结果显示,微量法能在较短时间内获取开环较少的闭合环状鼠疫菌质粒DNA,经琼脂糖凝胶电泳图示其电泳条带清晰、亮度均一.微量法鼠疫菌质粒DNA抽提效率和纯度较好,抽提结果稳定,重复性良好.经微量法抽提的质粒DNA符合多数鼠疫菌分子生物学试验的要求,可广泛应用于鼠疫菌分子生物学试验研究中.     

Influence of neutrophilokines on the synthesis of lymphokines in the process of the formation of immunity to plague

The evaluation of the complex of neutrophilokines whose synthesis was induced by Yersinia pestis vaccine strain EV on the production of lymphokines in the process of the formation of primary and secondary immunity to plague is presented. As revealed in this study, neutrophilokines regulate the synthesis of IL-2 by T helpers of type 1, IL-4 and IL-5 by T helpers of type 2, IL-1 by B lymphocytes, as well as the expression of receptors IL-2 by immunocompetent cells. The helper effect of neutrophilokines is more pronounced in the secondary immune response.     

Yersinia pestis L-forms in rodents and ectoparasites

Y. pestis L-forms and bacterial forms persist in the body of great gerbils for 40 days. L-forms are poorly phagocytized and can persist in phagocytes for a long time. In guinea pigs immunized with vaccine EV, Y. pestis antigen could be detected till day 160. An unstable L-form was isolated from Ornithodoros mites 3 years after their experimental infection with Y. pestis. Bacterial forms persist in mites for 1-3 years. For 5 years Y. pestis antigen is regularly detected in a high percentage of mites.     

Immunochemical study of heterogenous antigens Y. pestis EV similar to human red cells]

Franctions containing heterogenous antigens Y. pestis EV similar to human red cells can be obtained by the method of immunosorption of antigens by fixed antibodies on polyacrylamide gel.     

The colonization of plants by Yersinia pestis EV in an experiment

The penetration of Y. pestis (strain EV) into the stem of Impatiens walleriana via its roots, submerged into microbial suspension, was observed under experimental conditions. This was indicative of the colonization of plants by Y. pestis, thus confirming the possibility of their preservation in plants during periods between epizootics at the territories of natural foci.     

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.