EHRLICHIACIDAL MECHANISM BY MACROPHAGE CA++ MOBILIZATION

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Ehrlichiae cause diseases such as human ehrlichiosis and sennetsu ehrlichiosis. Ehrlichiae are a unique group of rickettsiae and are obligate intracellular parasites of macrophages or granulocytes. They multiply in the membrane-bound vacuoles which do not fuse with lysosomes. The goal of this project is to find whether macrophage activating agents kill ehrlichiae and if so how ehrlichiae can be completely eliminated from the host cells. We will test the following specific hypotheses. Hypothesis 1: Ehrlichial infection blocks Ca2+ influx into the macrophage, thus the agents which can mobilize Ca 2+, in infected macrophages kill ehrlichiae. AIM 1: Tests hypothesis 1: a) by examining dose- and time- responses of macrophages in eliminating ehrlichiae in response to these agents, b) by measuring intracellular Ca2+ ion, and c) by examining the effects of Ca2+ channel blockers or Ca2+ antagonists. Hypothesis 2: Ehrlichial killing induced by Ca2+ mobilization is mediated by protein kinase C, calmodulin, phospholipase A2 and/or cAMP, or cGMP. AIM 2: Tests hypothesis 2 by a) evaluating the effects of protein kinase C inhibitors, calmodulin antagonists, Ca2+-dependent phospholipase A2 inhibitors, and agents which increase cAMP and cGMP on the ehrlichiacidal effects of Ca 2+ mobilizing agents, and b) measuring phosphodiesterase, protein kinase C, phospholipase A2 activity and/or cAMP and cGMP. Hypothesis 3: Ca2+ mobilization in the macrophage destroys ehrlichiae by initiation of lysosomal fusion with ehrlichia-containing vacuoles which is accompanied by increased polymerization and reorganization of tubulin and actin in the infected macrophage cytoplasm. AIM 3: Tests hypothesis 3 by a) examining the fusion of lysosomes labeled with electron densetracers or acid phosphatase cytochemistry with ehrlichiae-containing vacuoles, by b) fluorescent labeling of microfilaments and microtubules, and by c) examining the effect of microtubule and microfilament disrupting agents.