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

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.

FIG. 1

Schematic of L. pneumophila strain 130b open reading frames (indicated by arrows) in the lspDE (A) and lspFGHIJK (B) loci. For construction of the lspDE mutant, an internal 0.8-kb EcoRV fragment was deleted and replaced by a Km^r cassette (A). For the lspG mutant, the Km^r cassette was inserted in the NcoI site in the lspG open reading frame (B). Note that there is no NcoI site in lspH from L. pneumophila strain 130b, unlike in L. pneumophila strain Philadelphia-1 (32). The binding locations of the primers OR5, OR6, OR1, and OR2 used for amplification of the lsp probes are indicated by the small bars. Lower right bar, scale.

FIG. 2

Colony morphology of L. pneumophila strains. Wild-type (wt) 130b (A), lspDE mutant NU258 (B), lspG mutant NU259 (C), pilD mutant NU243 (D), and pilE_L mutant BS100 (E) were grown on BCYE agar at 37°C for 3 days and subsequently at room temperature for 9 days. Note that it may be difficult to see the cavity of the NU243 colonies in panel B. Although it might appear from the images in panels A to E that the strains' colonies had different sizes, this was not the case; e.g., panel F presents an image of three plates containing either 130b (upper left), NU243 (right) or NU258 (lower left).

FIG. 3

Secreted enzymatic activities of L. pneumophila strains. Culture supernatants of wild-type (wt) 130b, pilD mutant NU243, lspDE mutant NU258, lspG mutant NU259, and pilE_L mutant BS100 were tested for protease (A), acid phosphatase (B), p-NPPC hydrolase (C), lipase (D), phospholipase A (E), and lysophospholipase A (F) activities. These data represent the mean and standard deviation (error bars) for duplicate cultures. For all, the differences between the wild type and the pilD and lsp mutants were significant (P < 0.01 [Student's t test]). Comparable results were obtained on at least two other occasions (data not shown).

FIG. 4

Intracellular infection of amoebae with L. pneumophila strains. Wells containing H. vermiformis were infected at a multiplicity of infection of 0.1 with 130b (black squares), NU243 (white diamonds), NU258 (white squares), or NU259 (white triangles). Bacterial CFU per well were determined at 0, 24, and 48 h after inoculation. Each datum point represents the mean and standard deviation (error bars) of three wells. Significant differences in recovery between 130b and its mutant derivatives were evident at 48 h (P < 0.01 [Student's t test]). These differences were observed in three additional experiments (data not shown).

FIG. 5

Macrophage infection by L. pneumophila strains. (A) U937 cells were infected at a multiplicity of infection of 0.1 with 130b (black squares), NU243 (white diamonds), NU258 (white squares), or NU259 (white triangles). Bacterial CFU per monolayer were determined at 0, 24, and 48 h after inoculation. Each datum point represents the mean and standard deviation (error bars) of three wells. Significant differences in recovery between 130b and its lsp mutant derivatives were evident at 48 h (P < 0.001 [Student's t test]) and were observed in three additional experiments (data not shown). (B) Replicate U937 cell monolayers (n = 6) were either not infected or infected at a multiplicity of infection of 1 with wild-type 130b (black bars), pilD mutant NU243 (white bars), lspDE mutant NU258 (horizontally striped bars), or lspG mutant NU259 (wavy bars). After 16 and 42 h of incubation, the viability of the host cells was measured by their ability to reduce alamar blue. Datum points represent the mean and standard deviation (error bars) of the percentage of dye reduction by infected cells compared to uninfected cells. Significant differences in cytopathic effect between 130b and its mutant derivatives were evident at 42 h (P < 0.001 [Student's t test]). Similar results were obtained in two additional experiments, which used either a multiplicity of infection of 1 or 0.1 (data not shown).