Involvement of Ca2+ signaling in intracellular invasion of non-phagocytic host cells by Orientia tsutsugamushi

Calcium signaling has been implicated in various steps in bacterial pathogenesis. Here, we investigated the role of Ca2+ signaling in intracellular invasion of non-phagocytic host cells infected with Orientia tsutsugamushi, the causative agent of scrub typhus. The bacteria induced a transient Ca2+ increase in HeLa cells immediately after infection and the source of the mobilized Ca2+ appears to be intracellular stores, not the extracellular milieu, as determined using extracellular (EGTA) or intracellular (BAPTA-AM) Ca2+ chelators. O. tsutsugamushi rapidly induced activation of PLC-γ1, as indicated by tyrosine phosphorylation. PLC-γ1 activity increased within 1 min of infection and returned to the basal level by 5 min. Pretreatment of host cells with inhibitors of PLC-γ1 (U73122) or IP3R channel activity (2-APB) significantly blocked bacterial entry without affecting bacterial attachment. In addition, these chemical inhibitors were effective in suppressing not only the activation of ERK MAP kinase but also the expression of the chemokine MCP-1. Taken together, Ca2+ signaling induced by O. tsutsugamushi may play a crucial role in bacterial pathogenesis including inflammatory reactions as well as intracellular invasion into non-phagocytic host cells.