[POSSIBILITIES OF APPLICATION OF MALDI-TOF MASS-SPECTROMETRY FOR STUDY OF CARBOHYDRATE-SPECIFIC RECEPTORS FOR DIAGNOSTIC BACTERIOPHAGE EL TOR].

AIM: Study mechanisms of interaction of diagnostic bacteriophage El Tor with sensitive strain Vibrio cholerae El Tor 18507 using direct protein profiling, identification of constant and variable proteins, taking part in interaction of the phage and cell, as well as carbohydrate-specific phage receptors. MATERIALS AND METHODS: . A commercial preparation of cholera diagnostic bacteriophage El Tor, strain V. cholerae El Tor 18507 were used. Effect of carbohydrates on bacteriophage activity was determined in experiments with phage by a classic and modified by us method. Protein profiles of the studied objects were studied using MSP-analysis method. RESULTS: Sucrose was shown to inhibit lytic activity of bacteriophage. Proteome profiles of El Tor bacteriophage and sensitive indicator strains were studied, identification of constant and variable proteins of the studied objects by MSP Peak-list program was carried out. CONCLUSION: Analysis of changes of profiles of phage and microbial cell during interaction with sucrose gave a basis for assuming, that sucrose in the mixture of culture-phage enters interaction namely with phage protein receptors, blocking receptors specific for cholera vibrio, that subsequently manifests in a sharp decrease of phage activity against the sensitive strain.

[The mass-spectrometric analysis of MALDI-TOF in identification and typing of strains of comma bacillus].

The data base "Protein profiles of mass-specters of representatives of species of Vibrio cholerae for program MALDI Biotyper" was used to implement typing of strains of comma bacillus isolated at the territory of the Russian Federation in 2010-2012. Also, analysis of degree of similarity and differences among constant ribosomal proteins was implemented. According the results of MALDI-TOF mass-spectrometry strains of V.cholerae were grouped in two distinct clusters. The first cluster included all epidemically dangerous strains isolated from people arrived in Moscow from India 2010-2012. The second cluster included atoxigenic vibrio with no relation to serogroups O1/O139 isolated from residents of Taganrog in 2011. The analysis of main specters of all collection permitted to identify taxon - specific components distinguishing strains of non-O1/non-O139 from strains of V.cholerae El Tor. Hence, the developed data base of proteom portraits of V.cholerae permits identifying, studying and to typing of agents of cholera and other representatives of V.cholerae species detecting their phylogenetic affinity that is ultimately useful for establishing origin of strains isolated from objects of environment and epidemiological decoding of episodes of disease.

[Proteome mass-spectrometric analysis and typing of Vibrio cholerae strains isolated in the territory of Russian Federation in 2010 - 2012].

AIM: Conduction of a comparative proteomic mass-spectrometric (MS) analysis using a personal database of V. cholerae protein mass-spectra and genetic VNTR-typing of cholera causative agent strains. MATERIALS AND METHODS: V. cholerae O1 El Tor strains - 7, V. cholerae non O1/non O139 - 2. Protein profiling and VNTR-genotyping of strains was carried out on MALDI TOF-MS Autoflex (Bruker Daltonics) mass-spectrometer and SIS Cholera-strains-VNTR. RESULTS: The established community of a proteomic profile of epidemic cholera vibrio strains isolated in 2010 - 2012 in Moscow allowed to determine Indian origin of a toxigenic strain isolated in Taganrog in 2011. M/z proteins distinguishing V. cholerae O1 and non O1/non O139 strains were identified. Proteomic analysis confirms the results of VNTR-genotyping. CONCLUSION: Study and typing of V. cholerae members with determination of their origin and phylogenetic relationship is possible using a collection of V. cholerae mass-spectra.

[Role of galactose-specific receptor--lectin in bactericidal activity of hemolysin of Vibrio cholerae non O1/O139].

AIM: To study the role lectin galactose-specific receptor of Vibrio cholerae hemolysin in receptor mechanisms of bacterial cells lysis. MATERIALS AND METHODS: Five strains of V. cholerae eltor ctx- Hly+ and 5 strains V. cholerae eltorctx+ Hly(-) isolated from patients and environment as well as 6 strains from Shigella genus, 3 strains of Escherichia coli, 2 strains of V. cholerae eltor, and 1 strain of V. cholerae non O1 were used in the study. Preparation P-11702 obtained from strain of V. cholerae non O1 was used for the study of bactericidal activity of hemolysin. For neutralization of bactericidal effect antihemolytic serum prepared against P-11702 as well as 1% solutions of carbohydrates (galactose, sucrose, glucose, and N-acetyl-D-galactosamine) were used. RESULTS: It was shown that lytic activity of hemolysin against cholera vibrios and indicator cultures for detection of vibriocins was neutralized by galactose as well as hemolysis of erythrocytes was neutralized by specific antihemolytic serum. Sucrose, glucose, and N-acetyl-D-galactosamine did not have effect on bactericidal and hemolytic activity of hemolysin. CONCLUSION: Protein-carbohydrate receptor binding of galactose-specific lectin has an important role in realization of bactericidal effect of V. cholerae hemolysin.

[Search for primers on the basis of Yersinia pestis chromosomal DNA for effective PCR identification of typical and atypical plague pathogen strains].

The authors present the results of a comparative appraisal of a set of species-specific primers in the polymerase chain reaction (PCR) identification of typical and atypical plague pathogen strains. A hundred and seventy-three strains with a species specificity of Y. pestis and 67 heterologous strains were used to appraise the well-known Y. pestis chromosomal DNA-based primers: "3a", "yp2769ms06", "vlml2for/ISrev216", "vlm33/ISfor1754", as well as "JS" specific to Y. pseudotuberculosis". In some plague bacterial strains, the DNA sequences complementary to the primers "3a" and "yp2769ms06" were not found. All conceivable plague pathogen strains were detectable only with the primers "vlml2for/ISrev216" and "vlm33/ISfor1754". The primers "JS" recognized only the strains of pseudotuberculosis causative agent. The DNA of other microorganisms failed to react with none species of primers. For the effective detection and differentiation of typical and atypical Y. pestis strains, the authors propose PCR using a few pairs of primers with the merits of the primers of the group "vlm" and 'JS".