Role of Ca2+ and calmodulin in ehrlichial infection in macrophages.
Replication of Ehrlichia risticii was inhibited in P388D1 cells and murine peritoneal macrophages when a calmodulin antagonist (W-7, chlorpromazine, or trifluoperazine); a Ca2+ channel blocker (verapamil, diltiazem, nifedipine, or flunarizine); an extracellular Ca2+ chelator, EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]; an inhibitor of intracellular Ca2+ mobilization, TMB-8; or Ca2+ ionophore A23187 was added after internalization of the organism at 3 h postincubation. When intracellular ehrlichiae at their logarithmic stage of growth were treated with these reagents, not only was further proliferation prevented but also there was significant reduction in numbers of intracellular ehrlichiae. These reagents prevented spreading of E. risticii from P388D1 cells to THP-1 cells. None of these reagents prevented binding of [35S]methionine-labeled E. risticii to P388D1 cells, but all of these reagents prevented internalization of [35S]methionine-labeled E. risticii. Protein kinase C inhibitors, H-7 and staurosporin, had no effect. 14CO2 production from L-[14C]glutamine in Percoll-density-gradient-purified E. risticii was inhibited by A23187 but not by W-7 or verapamil, suggesting that Ca2+ but not calmodulin directly regulates ehrlichials glutamine oxidation. Pretreatment of E. risticii with W-7 or verapamil did not reduce its infectivity. These results indicate that calmodulin and Ca2+ are essential for ehrlichial internalization, replication, and spreading in macrophages but are not essential for binding.
Rikihisa
Y
Y
Department of Veterinary Biosciences, College of Veterinary Medicine, Ohio State University, Columbus 43210, USA.
Zhang
Y
Y
Park
J
J
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002147:Calmodulin / Q000502:physiology*; D004536:Ehrlichia / Q000187:drug effects / Q000502:physiology*; D004734:Energy Metabolism / Q000187:drug effects; D005260:Female; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D009573:Nitrites / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002121:Calcium Channel Blockers; D002147:Calmodulin; D009573:Nitrites; D013449:Sulfonamides; D000001:Calcimycin; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; C017967:W 7; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium