Type II protein secretion is a subset of the PilD-dependent processes thatfacilitate intracellular infection by Legionella pneumophila

Previously, we had demonstrated that a Legionella pneumophila prepilin pept idase (pilD) mutant does not produce type IV pill and shows reduced secreti on of enzymatic activities. Moreover, it displays a distinct colony morphol ogy and a dramatic reduction in intracellular growth within amoebae and mac rophages, two phenotypes that are not exhibited by a pilin (pilE(L)) mutant . To determine whether these pilD-dependent defects were linked to type II secretion, we have constructed two new mutants of L. pneumophila strain 130 b. Mutations were introduced into either lspDE, which encodes the type II o uter membrane secretin and ATPase, or lspFGHIJK, which encodes the pseudopi lins. Unlike the wild-type and pilE(L) strains, both IspDE and IspG mutants showed reduced secretion of six pilD-dependent enzymatic activities; i.e., protease, acid phosphatase, p-nitrophenol phosphorylcholine hydrolase, lip ase, phospholipase A, and lysophospholipase A. However, they exhibited a co lony morphology different from that of the pilD mutant, suggesting that the ir surfaces are distinct. The pilD, IspDE, and IspG mutants were similarly and greatly impaired for growth within Hartmannella vermiformis, indicating that the intracellular defect of the peptidase mutant in amoebae is explai ned by the loss of type II secretion. When assessed for infection of U937 m acrophages, both lsp mutants exhibited a 10-fold reduction in intracellular multiplication and a diminished cytopathic effect. Interestingly, the pilD mutant was clearly 100-fold more defective than the type II secretion muta nts in U937 cells. These results suggest the existence of a novel pilD-depe ndent mechanism for promoting L. pneumophila intracellular infection of hum an cells.