Inhibitory mechanism of human neutrophil apoptosis by Anaplasma phagocytophilum and identification of novel surface proteins of A. phagocytophilum and Ehrlichia chaffeensis

by Ge, Yan.

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 (an anti-apoptotic bcl-2 family member) mRNA expression and activation of caspase 3 (the main executioner caspase) 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. This suggests that A. phagocytophilum inhibits the intrinsic pathway of the spontaneous neutrophil apoptosis by protecting the mitochondrial membrane integrity. 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. In the extrinsic pathway of spontaneous neutrophil apoptosis as well as anti-Fas-induced neutrophil apoptosis, the activation of ii caspase 8 (initiator caspase of extrinsic pathway) and pro-apoptotic Bid (linking the intrinsic and extrinsic pathways) 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. However, with the exception of P44 outer membrane protein family, the surface components of A. phagocytophilum are largely unknown. To globally identify the major surface proteins of A. phagocytophilum, a membrane-impermeable biotin reagent, Sulfo-NHS-SS- Biotin, 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, hypothetical protein APH_0404, hypothetical protein APH_0405, P44 family proteins and OMP-1A. APH_0404 and APH_0405 were highly conserved in the family Anaplasmataceae. Except P44s, all of them were newly identified as major surfaceexposed proteins. Ehrlichia chaffeensis belongs to the same family of Anaplasmataceae as A. phagocytophilum. Despite the importance of surface proteins as a crucial interface for E. chaffeensis-host interactions, the knowledge of them is limited. So far, only P28, gp47 and gp120 have been shown as surface-exposed proteins. To globally investigate surface proteins of E. chaffeensis, Sulfo-NHS-SS-Biotin-streptavidin affinity-captured surface proteins were subjected to Nano-LC/MS/MS analysis. Nineteen out of 22 OMP- 1/P28 family proteins were detected in E. chaffeensis cultured in human monocytic iii leukemia THP-1 cells. For the first time, seventeen OMP-1/P28 family proteins were demonstrated to be expressed at the protein level. In addition, OMP85, hypothetical protein ECH_0525, gp47 and 11 other proteins were detected. 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. iv