BIOLOGICAL ACTIVITY OF YERSINIA PSEUDOTUBERCULOSIS TOXINS

Aim. Study of effect of heat-labile (HLT) and thermostable (HST) lethal toxins of Yersinia pseudotuberculosis on the development of embryos of sea urchin Strongylocentrotus intermedius, processes of biosynthesis of nucleic acids and protein in embryo cells and activity of nucleoside-kinases of sea urchin. Materials and methods. Y. pseudotuberculosis strains 2517 (pYV-) and 512 (pYV48MD, pYV82MD) were used for isolation of HLT and HST. Gametes and embryos of sea urchin S. intermedius were used to carry out the experiments and isolate nucleoside-kinases. Results. Both of the studied toxins of Y. pseudotuberculosis possessed spermiotoxic effect and reduced fertilizing ability of sea urchin spermies. HLT LD50 was 1 (ig/ml, and HST - 2 pg/ml. Toxins affected the development of embryos of sea urchin resulting in severe morphologic damages, cessation of the development of embryos at early stages of embryogenesis, destruction of cells and death of embryos. Wherein, damaging effect of HLT was observed at lower concentrations compared with HST. HLT inhibited DNA and RNA biosynthesis at concentrations of 1-2 pg/ml. HST did not affect biosynthesis of nucleic acids even at high concentrations, but inhibited protein biosynthesis in sea urchin embryos. HLT did not reduce the level of inclusion of labeled amino acids into embryo cells. HLT had inhibiting effect on the activity of thymidine- and uridine-kinase of sea urchin, whereas HST did not affect these enzymes. Conclusion. Both of Y. pseudotuberculosis protein toxins affect the development of sea urchin embryos, however, mechanisms of action of HLT and HST on embryos and processes occurring in them differ.</jats:p

BIOLOGICAL ACTIVITY OF YERSINIA PSEUDOTUBERCULOSIS TOXINS

Aim. Study of effect of heat-labile (HLT) and thermostable (HST) lethal toxins of Yersinia pseudotuberculosis on the development of embryos of sea urchin Strongylocentrotus intermedius, processes of biosynthesis of nucleic acids and protein in embryo cells and activity of nucleoside-kinases of sea urchin. Materials and methods. Y. pseudotuberculosis strains 2517 (pYV-) and 512 (pYV48MD, pYV82MD) were used for isolation of HLT and HST. Gametes and embryos of sea urchin S. intermedius were used to carry out the experiments and isolate nucleoside-kinases. Results. Both of the studied toxins of Y. pseudotuberculosis possessed spermiotoxic effect and reduced fertilizing ability of sea urchin spermies. HLT LD50 was 1 (ig/ml, and HST - 2 pg/ml. Toxins affected the development of embryos of sea urchin resulting in severe morphologic damages, cessation of the development of embryos at early stages of embryogenesis, destruction of cells and death of embryos. Wherein, damaging effect of HLT was observed at lower concentrations compared with HST. HLT inhibited DNA and RNA biosynthesis at concentrations of 1-2 pg/ml. HST did not affect biosynthesis of nucleic acids even at high concentrations, but inhibited protein biosynthesis in sea urchin embryos. HLT did not reduce the level of inclusion of labeled amino acids into embryo cells. HLT had inhibiting effect on the activity of thymidine- and uridine-kinase of sea urchin, whereas HST did not affect these enzymes. Conclusion. Both of Y. pseudotuberculosis protein toxins affect the development of sea urchin embryos, however, mechanisms of action of HLT and HST on embryos and processes occurring in them differ

EFFECT OF YERSINIA PSEUDOTUBERCULOSIS TOXINS ON THE BIOFILM FORMATION

Aim. To study the effect of heat-labile (HLTY) and heat-stable (HSTY) lethal toxins of the of Yersinia pseudotuberculosis on the formation of biofilms by these bacteria. Materials and methods. For the isolation of toxins and the investigation their ability to effect on the biofilm formation there were used the strain of Y. pseudotuberculosis 512 (pYV48Mjl. рУМ82МД) and strain 2517, carrying virulence plasmid pYV and lost it, correspondingly. Results. The stimulation of biofilm formation at 20°C by the strain 2517 (pYV+), carrying virulence plasmid as well as the strain 2517 (pYV-) without plasmid were observed In the presence of HLTY. At low positive temperature (6 - 8°C) HLTY reduces the amount of the formed biofilm. HSTY inhibited the biofilm formation by the both strains of Y. pseudotuberculosis tested during the incubation for 3 days at 20°C and 6 - 8°C. Moreover the extent of inhibition was decreased with temperature decreasing. Conclusion. The both of the Y. pseudotuberculosis protein toxins has been revealed to affect on the biofilm formation by Y. pseudotuberculosis bacteria, however, the impact of HLTY and HSTY in the processes of biofilm formation was shown to be different, and the mechanism of such action of toxinsis under way.</jats:p

Recombinant α-NAcetylgalactosaminidase from Marine Bacterium-Modifying A Erythrocyte Antigens

A plasmid based on pET-40b was constructed to synthesize recombinant -N-acetylgalactosaminidase of the marine bacterium Arenibacter latericius KMM 426 T (-AlNaGal) in Escherichia coli cells. The yield of -Al-NaGal attains 10 mg/ml with activity of 49.7 1.3 U at 16C, concentration of inductor 2 mM, and cultivation for 12 h. Techniques such as anion exchange, metal affinity and gel filtration chromatography to purify -AlNaGal were applied. -AlNaGal is a homodimer with a molecular weight of 164 kDa. This enzyme is stable at up to 50C with a temperature range optimum activity of 20-37C. Furthermore, its activity is independent of the presence of metal ions in the incubation medium. 1H NMR spectroscopy revealed that -AlNaGal catalyzes the hydrolysis of the O-glycosidic bond with retention of anomeric stereochemistry and possesses a mechanism of action identical to that of other glycoside hydrolases of the 109 family. -AlNaGal reduces the serological activity of A erythrocytes at pH 7.3. This property of -AlNaGal can potentially be used for enzymatic conversion of A and AB erythrocytes to blood group O erythrocytes.</jats:p

Stereochemical course of hydrolytic reaction catalyzed by alpha-galactosidase from cold adaptable marine bacterium of genus Pseudoalteromonas

The recombinant α-galactosidase of the marine bacterium (α-PsGal) was synthesized with the use of the plasmid 40Gal, consisting of plasmid pET-40b (+) (Novagen) and the gene corresponding to the open reading frame of the mature α-galactosidase of marine bacterium Pseudoalteromonas sp. KMM 701, transformed into the E. coli Rosetta(DE3) cells. In order to understand the mechanism of action, the stereochemistry of hydrolysis of 4-nitrophenyl α-D-galactopyranoside (4-NPGP) by α-PsGal was measured by 1H NMR spectroscopy. The kinetics of formation of α- and β-anomer of galactose showed that α-anomer initially formed and accumulated, and then an appreciable amount of β-anomer appeared as a result of mutarotation. The data clearly show that the enzymatic hydrolysis of 4-NPGP proceeds with the retention of anomeric configuration, probably, due to a double displacement mechanism of reaction