RESUMEN
BACKGROUND AND OBJECTIVES: The properties of neuraminidase produced by P. aeruginosa strain PAO1 during growth in a defined medium (BHI) was examined and compared with some neuraminidase features of K. pneumoniae in this investigation. MATERIALS AND METHODS: The enzyme was isolated from concentrated culture supernatants of P. aeruginosa which was used in a sensitive fluorometric assay by using 2'-(4-methylumbelliferyl) α-D-N acetylneuraminic acid as substrate. RESULTS: Neuraminidase production in P. aeruginosa PAO1 paralleled bacterial growth in defined medium (BHI) and was maximal in the late logarithmic phase of growth but decreased during the stationary phase, probably owing to protease production or thermal instability. Highest production of P. aeruginosa PAO1 neuraminidase was in BHI culture media. The neuraminidase of P. aeruginosa PAO1 possessed an optimum temperature of activity at 56°C and the activity was maximal at pH 5. Heating the enzyme to 56°C for 45 min., in the presence of bovine serum albumin destroyed 33.1% of it's activity and addition of Ca(+2), EDTA and NANA also decreased activity markedly. CONCLUSION: The results revealed that the highest specific activity is for p. aeruginosa PAO1.
RESUMEN
BACKGROUND AND OBJECTIVES: Glycoprotein 96 is the primary chaperone of the endoplasmic reticulum. Immunization with it induced potent Cytotoxic T lymphocyte responses to intracellular bacteria. S. typhimurium is a facultative intracellular bacterium and acquired resistance against this bacterium mainly depends on activity of Cytotoxic T cells. This study aimed to evaluate the capacity of Glycoprotein 96 rich lysate as a vaccine candidate to induce a protective immune response in mice against a lethal dose challenge with Salmonella typhimurium. MATERIALS AND METHODS: Mice were infected with S. typhimurium. Then their spleens and livers were harvested and homogenized and the protein content of whole crude lysate was enriched using ammonium sulfate precipitation. SDS-polyacrylamide gel electrophoresis transfer method was used for enrichment of the protein from crude sample. Immunoblotting was conducted to detect Glycoprotein 96. Isoelectric point was achieved through the use of isoelectric focusing. PBS and whole crude lysate (from uninfected and infected mice) were injected to mice of test group, mice of control-1 group and mice of control-2 group, respectively, on days 0 and 14. Twenty-one days after the last immunization, the LD50 and bacterial loads of livers and spleens were determined. RESULTS AND CONCLUSION: Immunization with Glycoprotein 96 rich lysate isolated from livers and spleens of S. typhimuriuminfected mice induced protection against infection by S. typhimurium. Also, the bacterial burden of livers and spleens in mice that received gp96 rich lysate significantly decreased when compared to that of mice in the control groups.