Crucial Role of TolC in the Inhibition of Cellular Trafficking in the Protistan Host .

is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires' disease. In the environment, this bacterium survives in free-living protists such as amoebae and . The association of and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of infection for humans. spp. are candidates of natural hosts of , but their detailed relationships remain unclear. In the present study, we used an environmental strain, Ofk308 (Ofk308) and st110-1a to reveal the relationship between and spp. Ofk308 was cytotoxic to in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of in Ofk308. We used a LysoTracker to evaluate the maturation process of phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the -deletion mutant. To assess the phagocytic activity of , Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by through a TolC-dependent mechanism. Further, we evaluated the excretion of -containing vacuoles from . We found that failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for to remain within cells and to show cytotoxicity. Because of the high mobility and high cell division rate of spp., living with spp. would be beneficial for to expand its habitat. To control Legionaries' disease, understanding the ecology of in the environment is essential.