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.

FIG. 1.

Intracellular infection of H. vermiformis amoebae and U937 cell macrophages by L. pneumophila pilD mutants and their complemented derivatives. (A) Wells containing H. vermiformis were inoculated at a multiplicity of infection (MOI) of 0.1 with strains 130b(pMMB2002) (⋄), 130b(pMD1) (♦), NU243(pMMB2002) (□), NU243(pMD1) (▪), NU272(pMMB2002) (○), and NU272 (pMD1) (•), and then the numbers of bacteria in each well were quantified at various times postinoculation by plating on BCYE agar. Results are the means and standard deviations (error bars) of four wells and are representative of two independent experiments. In a third experiment, NU243(pMD1) and NU272(pMD1) also showed full complementation at 72 h postinoculation. (B) U937 cells were infected at an MOI of 0.1 with the same strains indicated above. At various times, the monolayers were lysed, and the total number of CFU in each well was determined. Results are the means and standard deviations (error bars) of triplicate wells and are representative of two independent experiments. The differences in recovery between 130b(pMD1) and both NU243(pMD1) and NU272(pMMB2002) were significant at 24 and 48 h (Student's t test, P < 0.005). (C) Macrophages were infected at an MOI of 0.1 with wild-type 130b (♦), Km^r mutant NU243 (□), Gm^r pilD mutant NU272 (○), and lspG mutant NU259 (▵), and then at various times, the total number of CFU in each well was determined. Results are the means and standard deviations (error bars) of triplicate wells and are representative of three independent experiments. At 24 and 48 h postinoculation, significant differences in recovery were obtained between 130b and NU243, 130b and NU259, 130b and NU272, and NU243 and NU272 (Student's t test, P < 0.001).

FIG. 2.

Intracellular infection of H. vermiformis amoebae and U937 cell macrophages with an L. pneumophila lspF mutant and its complemented derivative. Amoebae (A) and U937 cells (B) were inoculated with strains 130b(pMMB2002) (⋄), 130b(pMF1) (♦), NU275(pMMB2002) (○) and NU275(pMF1) (•) at a multiplicity of infection of 0.1, and then the numbers of bacteria in each well were determined at various times postinoculation. For both panels, results are the means and standard deviations (error bars) of triplicate wells and are representative of two independent experiments. (B) The differences in CFU recovery between 130b(pMMB2002)- versus NU275(pMMB2002)-infected monolayers were significant at both 24 and 48 h postinoculation (Student's t test, P < 0.005).

FIG. 3.

Intracellular infection of H. vermiformis amoebae and U937 cell macrophages by L. pneumophila pilQ, pspA, and lsp mutants. Amoebae (A and B) and U937 cells (C and D) were infected at a multiplicity of infection of 0.1 with various strains, and then the numbers of bacteria in each well were determined at various times postinoculation. (A and C) Infection profiles for strains 130b (♦), pilQ mutant NU279 (□), lspDE mutant NU258 (•), and lspDE pilQ double mutant NU283 (▵). (B and D) Infectivities of strains 130b (♦), pspA mutant NU280 (○), and lspG mutant NU259 (▵). The results are the means and standard deviations (error bars) of triplicate wells and are representative of two independent experiments. In panels C and D, significant differences in recovery were obtained between 130b and NU258, 130b and NU283, and 130b and NU259 (Student's t test, P < 0.005).

FIG. 4.

In vivo competition between wild-type L. pneumophila and mutants lacking type II protein secretion and Tfp. A mixture of 130b and mutant were introduced into the lungs of A/J mice by intratracheal inoculation. At 1 day (A) and 3 days (B) postinoculation, the ratio of 130b to mutant in infected lungs was determined and normalized to the ratio of wild type to mutant in the inoculum. Data are representative of actual values obtained per mouse (n = 4 to 6), and the solid bars represent the mean values. Strains tested in competition with wild type were from left to right: lspDE mutant NU258 (black circles), lspF mutant NU275 (gray squares), pilE_L mutant BS100 (gray circles), pilQ mutant NU279 (white squares), and pilD mutant NU272 (white circles). For the trials done with NU275, BS100, NU279, and NU272, significant differences were obtained between ratios observed at day 1 and day 3 (Student's t test, P < 0.05).

FIG. 5.

In vivo competition between wild-type L. pneumophila and mutant NU243. A mixture of 130b and the Km^r mutant NU243 was introduced into the lungs of A/J mice by intratracheal inoculation. At 1 and 3 days postinoculation, the ratio of 130b to mutant in infected lungs was determined. Data are representative of the actual values obtained per mouse (n = 4), and a solid bar represents the mean value. The 130b to mutant ratio increased significantly between days 1 and 3 (Student's t test, P < 0.05).

FIG. 6.

Growth and survival of wild-type and lspF mutant L. pneumophila in the lungs of infected A/J mice. Mice were intratracheally inoculated with equal numbers of either 130b (black diamonds) or lspF mutant NU275 (gray squares), and then at various time points, the CFU in infected lungs were determined by plating. Data are the means and standard deviations (error bars) obtained for four to six infected animals. Significant differences were obtained between the CFU recovered from mice infected with 130b and those infected with NU275, at 48 and 96 h postinoculation (Student's t test, P < 0.01).

FIG. 7.

Immunodetection of L. pneumophila type II-secreted proteins by infected mouse sera. Concentrated supernatants from cultures of L. pneumophila strains 33155 (lane 1) and 130b (lane 2) and lspF mutant NU275 (lane 3) were separated by SDS-PAGE, transferred to a nitrocellulose membrane and reacted with the diluted serum of a 130b-infected A/J mouse. Similar results were obtained using the serum of a second infected mouse.