The research goal is to understand the mechanisms by which IFN-gamma and various Ca2+ modulating agents induce ehrlichial killing. Hypothesis 1 is that IFN-gamma-activated monocytes inhibit the multiplication of Ehrlichia spp. by limiting the availability of intracellular iron. Hypothesis 2 is that ehrlichial ATP synthesis is Ca2+ dependent. Hypothesis 3 is that calmodulin antagonists and Ca2+ channel blockers, by inhibiting clathrin assembly, block maintenance and addition of increments of inclusion membrane and transferrin receptor recycling. Hypothesis 4 is that all these inhibitions make ehrlichiae incapable of resisting acidification and lysosomal fusion with inclusion vacuoles. To prove these hypotheses, the specific research aims are:
Aim 1. Evaluate the inhibitory mechanism of E. chaffeensis and E. sennetsu in human monocytes by IFN- by examining: 1) the effects of dosages of IFN-gamma at different time points, 2) the numbers and affinities of transferrin receptors with or without IFN-gamma treatment and reversibility of the inhibitory effect with iron-saturated transferrin, 3) inhibitory effect of chloroquine and NH4Cl on ehrlichial proliferation, and 4) iron-uptake mechanism by Ehrlichia spp.
Aim 2. Study the effect of Ca2+ on Percoll-density gradient purified ehrlichiae by measuring: 1) 14C-L-glutamine metabolism and ATP production by ehrlichiae in the presence of different concentrations of Ca2+, and 2) Ca2+ concentration in ehrlichiae by electron probe X-ray microanalysis.
Aim 3. Study the inhibitory mechanism of ehrlichiae through inhibition of clathrin assembly by: 1) fluorescence double labeling of infected monocytes with anti- clathrin IgG and anti-Ehrlichia spp. IgG after treatment with W-7, verapamil, or monodansylcadaverine, 2) high-resolution electron microscopy of platinum replicas of freeze-dried fractured cells, and 3) examining whether iron nitrilotriacetates can reverse the inhibition.
Aim 4. Examine whether acidification and lysosomal fusion are involved in ehrlichial killing induced by all these reagents by: 1) immunofluorescence and immunogold labeling with anti-vacuolar type H+-ATPase antibody, 2) measuring accumulation of weak bases in the vacuole containing ehrlichiae, and 3) examining lysosomal fusion by acid phosphatase cytochemistry, electron-dense tracer ThO2-preloading and - chasing method, and immunofluorescence and immunogold labeling with anti- lysosomal glycoprotein antibody.
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