The novel Legionella pneumophila type II secretion substrate NttC contributes to infection of amoebae Hartmannella vermiformis and Willaertia magna

Abstract

The type II protein secretion (T2S) system of Legionella pneumophila secretes over 25 proteins, including novel proteins that have no similarity to proteins of known function. T2S is also critical for the ability of L. pneumophila to grow within its natural amoebal hosts, including Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria lovaniensis. Thus, T2S has an important role in the natural history of legionnaires' disease. Our previous work demonstrated that the novel T2S substrate NttA promotes intracellular infection of A. castellanii, whereas the secreted RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all promote infection of H. vermiformis and N. lovaniensis. In this study, we determined that another novel T2S substrate that is specific to Legionella, designated NttC, is unique in being required for intracellular infection of H. vermiformis but not for infection of N. lovaniensis or A. castellanii. Expanding our repertoire of amoebal hosts, we determined that Willaertia magna is susceptible to infection by L. pneumophila strains 130b, Philadelphia-1 and Paris. Furthermore, T2S and, more specifically, NttA, NttC and PlaC were required for infection of W. magna. Taken together, these data demonstrate that the T2S system of L. pneumophila is critical for infection of at least four types of aquatic amoebae and that the importance of the individual T2S substrates varies in a host cell-specific fashion. Finally, it is now clear that novel T2S-dependent proteins that are specific to the genus Legionella are particularly important for L. pneumophila infection of key, environmental hosts.

Fig. 1.

The nttC locus and expression of nttC during extracellular growth. (a) Depiction of the region of the 130b chromosome containing nttC. The horizontal arrows denote the locations, orientations and relative sizes of the L. pneumophila genes. The ‘lpw’ numbers placed within the arrows are ORF designations used in the database. The gene names and annotations appear under the arrows. Sizes of the genes (bp) are given above the arrows, and sizes of the intergenic regions are given between the arrows. (b) Expression of nttC during L. pneumophila growth in broth culture. After 130b was grown in BYE broth at 37 °C, qRT-PCR was used to assess the fold change in nttC transcript levels during early stationary and late stationary phases as compared with exponential (E) phase. The data are shown as mean±sd from triplicate cultures (n = 3) or RNA samples. Results are representative of two independent experiments. (c) Wild-type (WT) strain 130b and its nttC mutant derivative, NU425, were inoculated into BYE broth, and then the cultures were incubated at 37 °C with shaking. At various times post-inoculation, the extent of bacterial growth was monitored spectrophotometrically. The data points represent the mean and sd of triplicate cultures, and the results presented are representative of three independent experiments.

Fig. 2.

Infection of N. lovaniensis, H. vermiformis and A. castellanii by wild-type strain 130b, nttC mutants and a complemented nttC mutant. N. lovaniensis (a), H. vermiformis (b, c) and A. castellanii (d) were infected with WT 130b and the nttC mutant NU425. H. vermiformis was also infected with the nttC mutant NU426 (b), and both H. vermiformis and A. castellanii were also infected with a complemented nttC mutant (nttC⁻/nttC⁺) (c, d). At the indicated times, the numbers of c.f.u. from the infected monolayers were determined. Data are the means and sd for four infected wells and are representative of at least three independent experiments.

Fig. 3.

Infection of W. magna and other amoebae by L. pneumophila 130b. (a) W. magna, (b) H. vermiformis, (c) A. castellanii and (d) N. lovaniensis were infected with WT strain 130b at a bacteria to host cell ratio of 5, and at the indicated times, c.f.u. from the infected monolayers were determined. Data are the means and sd for four infected wells, and are representative of at least three independent experiments (a, b, d) and of two separate trials (c).

Fig. 4.

Infection of W. magna by various wild-type strains and a T2S mutant. (a) Amoebae were infected with WT strains 130b, Philadelphia-1 (Phil) and Paris (a) and with WT 130b, lspF mutant NU275 and the complemented lspF mutant (b), and then c.f.u. from the infected monolayers were determined. Data are the means and sd for four infected wells, and are representative of three independent experiments (a). The comparison between WT and the lspF mutant (b) is also representative of five trials, and examination of the complemented mutant was done twice with comparable results.

Fig. 5.

Infection of W. magna by various T2S substrate mutants. Amoebae were infected with WT 130b (a–c), plaC mutant NU367 and its complemented derivative (a), nttA mutant NU415 and its complemented derivative (b), and nttC mutant NU425 and its complement (c), and then at the indicated times, the numbers of c.f.u. from the infected monolayers were determined. Data are the means and sd for four infected wells. The comparison between WT and the mutants is representative of at least five trials, and examination of the complemented mutants was done twice with comparable results. *P<0.05, **P<0.01 compared with WT.