Ehrlichia chaffeensis and Anaplasma phagocytophilum cause emerging infectious diseases, human monocytic ehrlichiosis (HME) and human granulocytic ehrlichiosis (HGE), respectively. The overall goal of the proposed study is to identify ehrlichiacidal mechanisms in the host cells, by focusing host signaling events essential (not just parallel phenomena) for intracellular parasitism. Thus, mechanisms can be potentially exploited to prevent and treat these infections. During the previous funding period this project identified several novel signaling pathways and mechanisms essential for E. chaffeensis and A. phagocytophilum infection. Blocking the host signals effectively killed these bacteria without harming the host human leukocytes in vitro. In this renewal proposal we will: 1. Elucidate mechanisms by which TG, GPI-anchored proteins, and tyrosine phosphorylated proteins are required for E. chaffeensis and A. phagocytophilum by identifying proteins involved by MALDI-TOF analysis, the gene knockdown, and immunolabeling. 2. Evaluate influences of cholesterol levels on mouse models of HGE and HME, and influences of cholesterol derivatives from ticks on E. chaffeensis and A. phagocytophilum infectivity. 3. Elucidate the mechanism by which A. phagocytophilum and E. chaffeensis block host cell NADPH oxidase activation in a cell-free system and COS-phox cells. 4. Elucidate the mechanism by which A. phagocytophilum inhibits human neutrophil apoptosis by analysis of caspases, bcl-2 family, and the mitochondrial integrity, transcriptional regulation of bcl-2 family proteins. The information to be obtained from the proposed study will point to unique mechanisms to kill E. chaffeensis and A. phagocytophilum and contribute to our understanding on host factors and signals induced by these pathogens and are essential for the obligatory intracellular parasitism. The results may point to a potential target for new treatment and prevention of HME and HGE.