Infect Immun 69(4): 2092-2098

Type II Protein Secretion Is a Subset of the PilD-Dependent Processes That Facilitate Intracellular Infection by <em>Legionella pneumophila</em>

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, Illinois 60611

^{Corresponding author. Mailing address: Department of Microbiology and Immunology, Northwestern University Medical School, 320 East Superior St., Chicago, IL 60611. Phone: (312) 503-0385. Fax: (312) 503-1339. E-mail:}ude.nretsewhtron@ottoicnaic-n.

Received 2000 Oct 27; Revisions requested 2000 Dec 1; Accepted 2000 Dec 20.

Abstract

Previously, we had demonstrated that a Legionella pneumophila prepilin peptidase (pilD) mutant does not produce type IV pili and shows reduced secretion of enzymatic activities. Moreover, it displays a distinct colony morphology and a dramatic reduction in intracellular growth within amoebae and macrophages, 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 130b. Mutations were introduced into either lspDE, which encodes the type II outer membrane secretin and ATPase, or lspFGHIJK, which encodes the pseudopilins. Unlike the wild-type and pilE_L strains, both lspDE and lspG mutants showed reduced secretion of six pilD-dependent enzymatic activities; i.e., protease, acid phosphatase, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A. However, they exhibited a colony morphology different from that of the pilD mutant, suggesting that their surfaces are distinct. The pilD, lspDE, and lspG mutants were similarly and greatly impaired for growth within Hartmannella vermiformis, indicating that the intracellular defect of the peptidase mutant in amoebae is explained by the loss of type II secretion. When assessed for infection of U937 macrophages, 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 mutants in U937 cells. These results suggest the existence of a novel pilD-dependent mechanism for promoting L. pneumophila intracellular infection of human cells.

Abstract

The gram-negative bacterium Legionella pneumophila is the agent of Legionnaires' disease, a potentially fatal pneumonia (14, 66). In nature, the organism replicates within protozoan hosts and biofilms found in aquatic environments (8, 16, 24). Following inhalation of aerosolized droplets, L. pneumophila invades and multiplies within alveolar macrophages (1, 57, 60, 64, 66). Various factors that are associated with L. pneumophila infection of protozoa and macrophages have been reported. These include major outer membrane proteins (29, 37), Mip (18, 27), flagella (49), type IV pili (58), a catalase-peroxidase (12), growth phase (17), iron acquisition (63), and the Dot-Icm putative type IV secretion apparatus (52, 55, 64).

Our previous studies have shown that a prepilin peptidase gene, pilD, is essential for Legionella growth in amoebae and human macrophages (38, 39). Moreover, a pilD mutant is dramatically reduced in virulence, following intratracheal inoculation of guinea pigs (38). By virtue of their ability to process pilin and the so-called pseudopilins, prepilin peptidases are implicated both in the formation of type IV pili and in protein secretion (41, 45, 46, 48, 59). One set of pseudopilins is involved in the assembly of the pili, and another one is involved in the genesis of a functional type II protein secretion system. Accordingly, the L. pneumophila pilD mutant does not produce type IV pili and lacks a number of secreted proteins and enzymatic activities in its culture supernatants (6, 28, 38). Interestingly, the pilD mutant also displays an altered colony morphology that is not associated with simple loss of pili (38). Since a mutant containing an insertion in the type IV pilin structural gene (pilE_L) is not defective for growth within amoebae and macrophages (58), we postulated the existence of a type II secretion system in L. pneumophila and argued that some secreted proteins might be virulence factors necessary for intracellular replication (38, 39). Prior to that study (38), pilD and type II secretion had not been implicated in bacterial intracellular infection. Importantly, a subsequent study demonstrated the presence of genes, lspFGHIJK (for Legionella secretion pathway), encoding some conserved components of type II secretion systems in L. pneumophila (32).

Through mutational analysis, the present study provides a definite link between L. pneumophila type II secretion and colony morphology, all known pilD-dependent enzymatic activities, and multiple forms of intracellular infection. This genetic approach had two key aspects. First, we mutated two loci (lspDE and lspFGHIJK) since it is believed that bacterial PilD proteins do not influence all portions of the type II system equally (26, 45, 46, 50, 53). In one locus, lspD is predicted to encode the outer membrane secretin, and lspE is predicted to encode the ATPase of the system (26, 50, 53). There are no data to suggest that type II secretins or ATPase are directly cleaved by PilD. The other locus, lspFGHIJK, is predicted to encode the pseudopilins, which, in other bacteria, are directly processed by PilD (45, 46, 53). Second, we introduced these mutations into the virulent strain 130b. As a result, the lsp mutants could be directly compared to the 130b mutants deficient in pilD and pilE_L, permitting clear distinctions between the relative roles of pilD, pilin, and type II protein secretion.

ACKNOWLEDGMENTS

We thank Yousef Abu Kwaik for kindly providing strain BS100.

This work was supported by National Institutes of Health grant AI43987 awarded to N.P.C.

ACKNOWLEDGMENTS

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