Inhibitory mechanism of human neutrophil apoptosis by anaplasma phagocytophilum and indentification of novel surface proteins of A. Phagocytophilum and ehrlichia chaffeensis
Abstract (Summary)The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signaling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. In this paper, it was found that the decrease of bfl-1 mRNA expression and activation of caspase 3 during spontaneous neutrophil apoptosis are inhibited by A. phagocytophilum infection. It was observed that most uninfected neutrophils lost mitochondrial membrane potential in contrast with high membrane potential in infected cells. Next, we studied the molecular signaling of the extrinsic apoptotic pathway (death receptor pathway) and the interaction between the intrinsic and extrinsic pathways in the inhibition of spontaneous human neutrophil apoptosis by A. phagocytophilum. Cell surface Fas clustering during spontaneous neutrophil apoptosis was significantly blocked by infection. The activation of caspase 8 and pro-apoptotic Bid were inhibited by A. phagocytophilum infection. These data together point to a novel mechanism induced by A. phagocytophilum involving both extrinsic and intrinsic pathways to ensure to delay the apoptosis of host neutrophils. Including inhibiting neutrophil apoptosis, the surface of A. phagocytophilum plays a crucial role in subverting the hostile host cell environment. To globally identify the major surface proteins of A. phagocytophilum, a membrane-impermeable biotin reagent was used to label the intact bacteria. Among the major proteins captured by biotin-streptavidin affinity purification, were five A. phagocytophilum proteins i.e., OMP85, two hypothetical proteins, P44 family proteins and OMP-1A. Except P44s, all of them were newly identified as major surface-exposed proteins. Ehrlichia chaffeensis belongs to the same family of Anaplasmataceae as A. phagocytophilum. So far, only P28, gp47 and gp120 have been shown as surface-exposed proteins. By using the affinity purification and proteomic methods, in addition to P28 and gp47, 31other proteins were detected in E. chaffeensis cultured in human monocytic leukemia THP-1 cells. The identification of E. chaffeensis surface-exposed proteins provides novel insights about E. chaffeensis surface and lays the foundation for rational studies on pathogen-host interaction and vaccine development.
School:The Ohio State University
School Location:USA - Ohio
Source Type:Master's Thesis
Date of Publication:01/01/2007