Inhibition of infection of macrophages with Ehrlichia risticii by cytochalasins, monodansylcadaverine, and taxol.
The requirement of functions of clathrin, microfilaments, and microtubules in binding, internalization, proliferation, and spreading of Ehrlichia risticii in macrophages was studied. Monodansylcadaverine (MDC), which inhibits clustering and internalization of the ligand-receptor complexes into clathrin-coated vesicles; cytochalasin B or D, which depolymerizes microfilaments; and taxol, which binds and stabilizes polymerized microtubules, were found to prevent ehrlichial infection in murine peritoneal macrophages when they were present throughout the infection period. [35S]methionine-labeled ehrlichial binding to the macrophage was reduced by 0 to 22%; therefore, the binding was not the major point of inhibition. However, MDC, cytochalasin D, and taxol inhibited ehrlichial internalization into macrophages by 80, 58, and 32%, respectively. When MDC, cytochalasin B or D, or taxol was added immediately after internalization of E. risticii (3 h postinfection), ehrlichial replication in P388D1 cells was almost completely prevented. Also, all of these agents almost completely prevented ehrlichial spreading from P388D1 cells to THP-1 human monocytes. These agents were not found to be ehrlichiacidal when approximately 40%-infected P388D1 cells were treated for 2 days, although further intracellular proliferation was prevented. Furthermore, none of these compounds directly inhibited the metabolic activity of E. risticii, since 14CO2 production from L-[14C]glutamine by Percoll density gradient-purified host-cell-free E. risticii was not shown to be impaired. The action of taxol was probably due to impairment of the microtubule function since a microtubule-depolymerizing agent, colchicine, also inhibited intracellular proliferation of E. risticii. Neither reactive oxygen intermediates, nitric oxide, nor tumor necrosis factor alpha appeared to be involved in taxol-induced inhibition of E. risticii in macrophages. Thus, our findings indicate that ehrlichial internalization appears to take place by a mechanism that is more dependent on the functions of clathrin and less dependent on the functions of microfilaments or microtubules. Replication within macrophages and intercellular spreading appear to require clathrin, microfilaments, and microtubules. Consequently, alteration of these structures with inhibitors can result in complete prevention of infection.
Rikihisa
Y
Y
Department of Veterinary Pathobiology, College of Veterinary Medicine, Ohio State University, Columbus 43210-1092.
Zhang
Y
Y
Park
J
J
D008841:Actin Cytoskeleton / Q000502:physiology*; D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002966:Clathrin / Q000502:physiology*; D003078:Colchicine / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003572:Cytochalasins / Q000494:pharmacology*; D004536:Ehrlichia / Q000254:growth & development*; D016873:Ehrlichiosis / Q000503:physiopathology*; D004705:Endocytosis / Q000187:drug effects; D005260:Female; D066298:In Vitro Techniques; D017737:Macrophages, Peritoneal / Q000382:microbiology*; D051379:Mice; D008870:Microtubules / Q000502:physiology*; D009573:Nitrites / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism
D002966:Clathrin; D003572:Cytochalasins; D009573:Nitrites; D017382:Reactive Oxygen Species; D003513:Cycloheximide; C008542:monodansylcadaverine; D002103:Cadaverine; D017239:Paclitaxel; D003078:Colchicine