ABSTRACT
A large number of bacterial pathogens have been identified as mediators of apoptosis in vitro and the induction of apoptosis might be an important step in the pathogenesis of these bacteria. In this study, we analyzed the interactions of Orientia tsutsugamuchi with J774 murine macrophage-like cells. The J774 cells were infected with Boryong strain of O. tsutsugamushi and the DNA was analyzed with agarose gel electrophoresis. We observed the typical laddering pattern of DNA fragmentation indicative of apoptosis in infected cells but not cells infected with heat-killed O. tsutsugamushi. We performed terminal deoxynucleotidyl transferase (TdT) assay to label the 3'-hydroxy ends of DNA breaks and observed intense brown staining of nuclei of infected macrophages. With Hoechst 33258 for staining nucleus, strong chromatin condensation was observed only in infected J774 cells. We also examined the cytokine secretion pattern of J774 cells during the rickettsial infection. The large amount of TNF-alpha and IL-10 were secreted after 24 hrs of infection, but the secretion of IL-1beta was increased in small amount. These results showed that O. tsutsugamushi induce apoptosis in murine macrophage-like cells by different mechanism from that of shigella which cause secretion of large amount of IL-1beta.
Subject(s)
Apoptosis , Bacteria , Bisbenzimidazole , Chromatin , DNA , DNA Breaks , DNA Fragmentation , DNA Nucleotidylexotransferase , Electrophoresis, Agar Gel , Interleukin-10 , Macrophages , Orientia tsutsugamushi , Shigella , Tumor Necrosis Factor-alphaABSTRACT
14-3-3 proteins are cytoplasmic proteins of about 29 kDa and have a minimum of seven isoforms. This protein is important in signal transduction with the ability of binding with phosphoserine of many signalling proteins. We expressed 14-3-3 protein tagged with 6 histidine residues in E. coli and purified the protein by nickel affinity chromatography. Using this purified protein as an antigen, we made rabbit antisera and mouse monoclonal antibodies to 14-3-3 zeta isoform. We subcloned cDNA of 14-3-3 zeta isoform derived from HeLa cell lamda gt 11 library into an E. coli expression vector which is designed to express heterologous protein with N- terminal 6 hidtidine tag. BALB/c mice were immunized with purified 14-3-3 protein and the hybridoma clones which produce monoclonal antibodies angainst 14-3-3 protein were selected. These monoclonal antibodies reacted with the recombinant protein expressed in E. coli as well as the 29-kDa native protein in various cell lines. However, they did not immunoprecipitate 14-3-3 protein. The monoclonal antibodies produced in this study can be valuable tools for the identification of the 14-3-3 in signal transduction study.