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Role of Ca2+ and calmodulin in ehrlichial infection in macrophages
1Department of Veterinary Biosciences, College of Veterinary Medicine, Ohio State University, Columbus 43210, USA.
Abstract:
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.
Insights
Calcium and calmodulin are crucial for Ehrlichia risticii infection of macrophages, controlling its entry, replication, and spread. Blocking these pathways inhibits bacterial growth but not initial cell binding.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Ehrlichia risticii is an obligate intracellular bacterium that causes important animal diseases.
- Understanding the host-pathogen interactions is key to developing effective treatments.
Purpose of the Study:
- To investigate the role of calcium (Ca2+) and calmodulin in the lifecycle of Ehrlichia risticii within host cells.
- To determine the specific stages of Ehrlichia risticii infection affected by calcium and calmodulin modulation.
Main Methods:
- Utilized various pharmacological agents including calmodulin antagonists, Ca2+ channel blockers, chelators, and ionophores.
- Assessed the effects of these agents on Ehrlichia risticii binding, internalization, replication, and spreading in macrophage cell lines (P388D1, THP-1).
- Measured bacterial metabolic activity (14CO2 production) and infectivity post-treatment.
Main Results:
- Calmodulin antagonists and Ca2+ modulators significantly inhibited Ehrlichia risticii internalization, replication, and cell-to-cell spreading.
- These agents reduced the number of intracellular bacteria and prevented spread to new cells.
- Binding of Ehrlichia risticii to host cells was not affected by the tested reagents.
- Ca2+ ionophore A23187 inhibited bacterial glutamine oxidation, suggesting direct regulation by Ca2+.
Conclusions:
- Calcium and calmodulin are essential for Ehrlichia risticii internalization, replication, and spreading in macrophages.
- Host cell calcium signaling pathways are critical targets for controlling Ehrlichia risticii infections.
- These findings provide insights into the host-pathogen interactions and potential therapeutic strategies.