Icm/Dot-dependent upregulation of phagocytosis by Legionella pneumophila

Legionella pneumophila is the causative agent of Legionnaires' disease, a s evere pneumonia. Dependent on the icm/dot loci, L. pneumophila survives and replicates in macrophages and amoebae within a specialized phagosome that does not fuse with lysosomes. Here, we report that phagocytosis of wild-typ e L. pneumophila is more efficient than uptake of icm/dot mutants. Compared with the wild-type strain JR32, about 10 times fewer icm/dot mutant bacter ia were recovered from HL-60 macrophages in a gentamicin protection assay. The defect in phagocytosis of the mutants could be complemented by supplyin g the corresponding genes on a plasmid. Using fluorescence microscopy and g reen fluorescent protein (GFP)-expressing strains, 10-20 times fewer icm/do t mutant bacteria were found to be internalized by HL-60 cells and human mo nocyte-derived macrophages (HMM Phi). Compared with icm/dot mutants, wild-t ype L.. pneumophila infected two to three times more macrophages and yielde d a population of highly infected host cells (15-70 bacteria per macrophage ) that was not observed with icm/dot mutant strains. Wild-type and icmT mut ant bacteria were found to adhere similarly and compete for binding to HMM Phi. In addition, wild-type L. pneumophila was also phagocytosed more effic iently by Acanthamoeba castellanii, indicating that the process is independ ent of adherence receptor(s). Wild-type L. pneumophila enhanced phagocytosi s of an icmT mutant strain in a synchronous co-infection, suggesting that i ncreased phagocytosis results from (a) secreted effector(s) acting in trans .