Legionella pneumophila Type II Protein Secretion Promotes Virulence in the A/J Mouse Model of Legionnaires' Disease Pneumonia

Abstract

Legionella pneumophila, the gram-negative agent of Legionnaires' disease, possesses type IV pili and a type II protein secretion (Lsp) system, both of which are dependent upon the PilD prepilin peptidase. By analyzing multiple pilD mutants and various types of Lsp mutants as well as performing trans-complementation of these mutants, we have confirmed that PilD and type II secretion genes are required for L. pneumophila infection of both amoebae and human macrophages. Based upon a complete analysis of lspDE, lspF, and lspG mutants, we found that the type II system controls the secretion of protease, RNase, lipase, phospholipase A, phospholipase C, lysophospholipase A, and tartrate-sensitive and tartrate-resistant acid phosphatase activities and influences the appearance of colonies. Examination of the developing L. pneumophila genome database indicated that the organism has two other loci (lspC and lspLM) that are predicted to promote secretion and thus a set of genes that is comparable to the type II secretion genes in other gram-negative bacteria. In contrast to lsp mutants, L. pneumophila pilus mutants lacking either the PilQ secretin, the PspA pseudopilin, or pilin were not defective for colonial growth, secreted activities, or intracellular replication. L. pneumophila dot/icm mutants were also not impaired for type II-dependent exoenzymes. Upon intratracheal inoculation into A/J mice, lspDE, lspF, and pilD mutants, but not pilus mutants, exhibited a reduced ability to grow in the lung, as measured by competition assays. The lspF mutant was also defective in an in vivo kinetic assay. Examination of infected mouse sera revealed that type II secreted proteins are expressed in vivo. Thus, the L. pneumophila Lsp system is a virulence factor and the only type II secretion system linked to intracellular infection.The gram-negative bacterium Legionella pneumophila is the agent of Legionnaires' disease, a pneumonia which especially affects immunocompromised individuals (28,89). An inhabitant of freshwater environments, L. pneumophila naturally replicates within protozoan hosts and in biofilms (27, 89). Following inhalation of contaminated aerosols, the bacterium reaches the human respiratory tract. Bacterial multiplication in alveolar macrophages is concomitant with cell death and damage to the lung tissue (89, 100).In gram-negative bacteria, the PilD prepilin peptidase is necessary for the cleavage and methylation of pilins and pseudopilins that assemble into type IV pili (Tfp) (55,68,69,87). In addition, PilD processes other pseudopilins that are necessary for the biogenesis of a functional type II protein secretion system (8,13,55,71,86). Accordingly, our previous mutational analysis determined that pilD is required for L. pneumophila piliation and protein secretion (4, 52). In L. pneumophila, Tfp promote attachment to host cells and are involved in competence for DNA transformation (84, 85). The L. pneumophila proteins believed to be secreted via the type II system include a zinc metalloprotease, acid phosphatase...

The Type II Secretion System of Legionella pneumophila Dampens the MyD88 and Toll-Like Receptor 2 Signaling Pathway in Infected Human Macrophages

“…1C). As we previously observed when infecting U937 cells (16,18), the T2S mutant displayed modestly impaired growth in THP-1 cells and PBMCs (see Fig. S1 in the supplemental material).…”

“…In mammalian hosts, T2S contributes to both intracellular infection of macrophages and the destruction of lung tissue. L. pneumophila lsp mutants that lack T2S have an ϳ10-fold reduction in intracellular growth in both U937 cells, a human macrophage-like cell line, and murine macrophages obtained from A/J mice (16)(17)(18). Data from our laboratory have also shown that this reduction in CFU is not due to an entry defect or increased degradation through the phagosome-lysosome pathway but is instead due to a replication defect in LCVs at 4 to 12 h postentry (19).…”

“…L. pneumophila strain 130b (ATCC BAA-74, also known as AA100) is a clinical isolate that served as our WT and parental strain for mutants (90). Both mutant strain NU275, which lacks a functional lspF gene that encodes an inner membrane component of the T2S apparatus, and mutant strain NU347, which lacks a functional flaA gene encoding flagellin, were described previously (18,91). A mutant lacking both flaA and lspF (NU430) was obtained by introducing pGlspF::Km (18) into NU347 by transformation (91) and then selecting for the acquisition of kanamycin resistance.…”

The role of protein secretion systems in the virulence of the intracellular pathogen Legionella pneumophila

“…A decrease in replication in both amoebae and macrophages was observed for single lspDE, lspG, lspK and lspF mutants. In addition, the important role of this secretion system in the virulence of L. pneumophila was confirmed because type II secretion was shown to play a role in the multiplication and survival of L. pneumophila in mammalian lungs (Rossier et al, 2004). Both type IV pili and the type II secretion system also play a role in the colonization and establishment of biofilms by L. pneumophila (Lucas et al, 2006 (Broich et al, 2006).…”

Molecular and functional characterization of type I signal peptidase from Legionella pneumophila

“…Rossier et al, 2004). Mutagenesis of lsp genes and the prepilin peptidase gene pilD revealed that type II secretion promotes the ability to infect both protozoan and macrophage hosts and to grow in the mammalian lung (Hales & Shuman, 1999;Liles et al, 1999;Rossier et al, 2004).…”