The role of the intracellular signaling pathway in Ehrlichia canis infection in vitro

Ehrlichia canis is a pleomorphic obligatory intracellular gram-negative bacterium that causes tick-borne canine ehrlichiosis. A systematic investigation on the pathogenesis of the disease has been hindered largely by lack of a reliable quantitative assay. Using quantitative real time PCR (qPCR), a quantitative assay for E. canis was developed and validated. Using the qPCR, the intracellular proliferation kinetics of E. canis in the canine 030F cell-line was studied for the first time.;Many intracellular bacterial pathogens have been shown to use Ca 2+ signaling in the host cells to induce their own internalization and proliferation. However, it is not clear how Ca2+ signaling influences internalization and proliferation of E. canis in the cytoplasm of eukaryotic cells. Thus, it was hypothesized that host's intracellular calcium signaling pathway plays an important role in internalization and proliferation of E. canis, and that the inhibition of the signaling pathway suppresses E. canis infection. This hypo-thesis was tested by evaluating the effect of the calcium signaling inhibitors on E. canis internalization and proliferation in 030F cell-line.;Ehrlichia canis internalization and proliferation were significantly suppressed by EGTA treatment. SKF-96365 and verapamil treatment resulted in increased E. canis internalization. Verapamil treatment suppressed E. canis proliferation. Chelation of intracellular Ca2+ by BAPTA/AM increased E. canis internalization and decreased E. canis proliferation. Inhibition of phospholipase C by neomycin and U-73122 significantly enhanced E. canis internalization. Ehrlichia canis proliferation was significantly suppressed by neomycin treatment, while it was unaffected by U-73122 treatment. 2-APB and thapsigargin treatment enhanced E. canis internalization while they significantly suppressed E. canis proliferation.;It was demonstrated that E. canis internalization was not associated with modulation of [Ca2+]i in the 030F cell-line by analyses utilizing confocal microscopy, flow cytometry and spectrofluorometry. The data were further confirmed by the inositol 1,4,5-triphosphate (IP3) assay, in which no significant IP3 concentration change related to E. canis load was detected.;It was concluded from the study that the host's Ca2+-dependent self defense mechanisms may play an important role in the pathogenesis of canine ehrlichiosis. Also, the 030F cell-line proved to be an efficient host for E. canis with predictably high (90-100%) susceptibility.;Keywords: Ehrlichia canis, quantitative real time PCR, calcium signaling, infection, in vitro cell culture system...