| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 94 === Oxidative stress plays important roles in many cellular states including bactericidal effect during phagocytosis. However, the bacterial killing mechanism by reactive oxygen species (ROS) remains obscure. Here, we investigated the role of bacterial type II topoisomerases (Topo IIs) in oxidative stress-mediated cell death. Hydrogen peroxide (H2O2) was used to induce oxidative stress in bacteria. The involvement of Topo II-mediated DNA damage in bactericidal activity during phagocytosis is supported by the following observations. (1) Cellular exposure of H2O2 led to activation of SOS response, an observation suggestive of DNA damage in E. coli. (2) H2O2 rapidly induced cleavage of nucleoid DNA into high-molecular-weight (HMW) DNA fragments in E. coli. (3) H2O2-induced HMW DNA fragments were covalently linked with proteins. (4) H2O2-induced HMW DNA fragmentations were highly reversible. (5) Coumermycin A1, a bacterial Topo II catalytic inhibitor, efficiently antagonized the formation of H2O2-induced HMW DNA fragments and the H2O2-induced SOS response. (6) Most importantly, coumermycin A1 attenuated the bacterial cell killing by H2O2 and bactericidal effect during phagocytosis in macrophage. Taken together, we concluded that H2O2 effectively targets bacterial Topo IIs and causes DNA breakage, which then leads to cell death. Our results therefore present the first demonstration that bacterial Topo IIs are the bactericidal target during phagocytosis.
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