Pore formation triggered by Legionella spp. is an Nlrc4 inflammasome-dependent host cell response that precedes pyroptosis

Abstract

Legionella pneumophila, the etiological agent of Legionnaires disease, is known to trigger pore formation in bone marrow-derived macrophages (BMMs) by mechanisms dependent on the type IVB secretion system known as Dot/Icm. Here, we used several mutants of L. pneumophila in combination with knockout mice to assess the host and bacterial factors involved in pore formation in BMMs. We found that regardless of Dot/Icm activity, pore formation does not occur in BMMs deficient in caspase-1 and Nlrc4/Ipaf. Pore formation was temporally associated with interleukin-1beta secretion and preceded host cell lysis and pyroptosis. Pore-forming ability was dependent on bacterial Dot/Icm but independent of several effector proteins, multiplication, and de novo protein synthesis. Flagellin, which is known to trigger the Nlrc4 inflammasome, was required for pore formation as flaA mutant bacteria failed to induce cell permeabilization. Accordingly, transfection of purified flagellin was sufficient to trigger pore formation independent of infection. By using 11 different Legionella species, we found robust pore formation in response to L. micdadei, L. bozemanii, L. gratiana, L. jordanis, and L. rubrilucens, and this trait correlated with flagellin expression by these species. Together, the results suggest that pore formation is neither L. pneumophila specific nor the result of membrane damage induced by Dot/Icm activity; instead, it is a highly coordinated host cell response dependent on host Nlrc4 and caspase-1 and on bacterial flagellin and type IV secretion system.

FIG. 1.

Caspase-1 is required for pore formation in response to Legionella infection. BMMs adherent to glass coverslips were infected with L. pneumophila at different MOIs for 1 h in the presence of anti-L. pneumophila antibody, and the cultures were processed for pore formation assays by double staining with EtBr and acridine orange. (A) Caspase-1^−/− and C57BL/6 BMMs were infected for 1 h with L. pneumophila at an MOI of 2, 10, or 100. (B) Representative images of pore formation in caspase-1^−/− or C57BL/6 BMMs at 1 h after infection with L. pneumophila at an MOI of 10 per cell. Acridine orange-stained BMMs (green) show total cells in each field, and EtBr (red) indicates permeabilized BMMs (middle panels). Right panels show merged images. Data are representative of those found in four independent experiments. *, P < 0.05 in comparison to C57BL/6 BMMs. WT, wild type.

FIG. 2.

Pore formation is temporally coordinated with IL-1β secretion and precedes host cell lysis. BMMs from caspase-1^−/− and C57BL/6 mice were treated with LPS for 4 h and infected with L. pneumophila at an MOI of 10 in the presence of anti-L. pneumophila antibody. Cultures were infected for 1, 2, and 3 h and processed for pore formation (A), release of LDH (B), or secretion of IL-1β (C). Data shown in all panels were obtained from the same cultures and are representative of three independent experiments. *, P < 0.05 in comparison to C57BL/6 BMMs.

FIG. 3.

Pore formation is dependent on the type IV secretion system but independent of bacterial multiplication and de novo protein synthesis. BMMs from C57BL/6 mice were infected with wild-type L. pneumophila (WT L.p.) or isogenic mutants for 1 h and assessed for pore formation. (A) BMMs were infected with GFP-expressing WT L. pneumophila or ΔdotA mutants at an MOI of 10 in the presence of anti-L. pneumophila antibody. Shown are merged images of GFP-expressing bacteria (green) and EtBr-positive nuclei (red). (B) BMMs were infected at an MOI of 100 of the wild-type strain and indicated isogenic mutants. ΔicmWS, ΔicmW ΔicmS. (C) BMMs were infected at an MOI of 100 of WT L. pneumophila in the absence (media) or presence of 20 μg/ml of chloramphenicol (Cm), or BMMs were infected with thyA and ΔdotA mutants. Data are representative three (A and B) or two (C) independent experiments. *, P < 0.05 in comparison to BMMs infected with WT L. pneumophila.

FIG. 4.

Pore formation is not a result of membrane damage by the Dot/Icm apparatus. BMMs from caspase-1^−/− and C57BL/6 mice were infected with wild-type (WT) L. pneumophila (WT L.p.) or isogenic ΔdotA mutants for 1 h at an MOI of 10 in the presence of anti-L. pneumophila antibody. Recruitment of ubiquitin (red) to L. pneumophila vacuoles (green) in caspase-1^−/− (A) or C57BL/6 (B) infected BMMs was visualized by confocal microscopy. Confocal high-resolution detail corresponds to ubiquitin recruitment to the vacuoles in the white inset. Scale bar, 10 μm. (C) Percentage of infected cells showing ubiquitin-positive L. pneumophila vacuoles upon infection with WT L. pneumophila or ΔdotA mutants. Data are representative of three independent experiments. *, P < 0.05 in comparison to BMMs infected with WT L. pneumophila.

FIG. 5.

Bacterial flagellin is sufficient to trigger pore formation. DOTAP was used to transfect proteins into BMMs obtained from C57BL/6 and caspase-1^−/− (casp-1^−/−) mice. Cultures were stimulated for 6 h and assayed for pore formation. (A) Cultures were treated with flagellin alone, DOTAP alone, DOTAP and p131 from S. mansoni, or DOTAP and flagellin. (B) BMM cultures were treated with DOTAP and a mock-flagellin preparation (extracted from ΔflaA L. pneumophila), DOTAP and flagellin, or DOTAP and flagellin treated with proteinase K. Data are representative of three independent experiments. *, P < 0.05 in comparison to BMMs treated with DOTAP and flagellin.

FIG. 6.

Flagellin expression is required for L. pneumophila induced pore formation. (A) BMMs from C57BL/6 mice were infected with wild-type L. pneumophila (WT L.p.) strain JR32 or ΔfliI, ΔflaA, or ΔdotA isogenic mutants at an MOI of 10 in the presence of anti-L. pneumophila antibody for 1 h and assessed for pore formation. (B) Bacteria grown on BCYE agar plates for 2 days were used to prepare extracts of total bacterial proteins. Extracts were separated by SDS-PAGE, blotted, and probed with antiflagellin antibody. Data are representative of three independent experiments. *, P < 0.05 in comparison to BMMs infected with WT L. pneumophila.

FIG. 7.

Nlrc4 is required for pore formation. BMMs obtained from C57BL/6, caspase-1^−/− (Casp-1^−/−), and Nlrc4^−/− mice were infected at an MOI of 2, 10, or 100 of the wild-type L. pneumophila (A) or ΔflaA mutant (B) strain for 1 h and assayed for pore formation. (C) Proteins from uninfected cultures (NI) or BMMs infected with wild-type L. pneumophila (WT L.p.) or the ΔdotA mutant at an MOI of 10 for 1 h were separated by SDS-PAGE, blotted, and probed with a caspase-1 antibody. Indicated at left are procaspase-1 (45 kDa) and subunit p20 of active caspase-1 (20 kDa). (D) BMMs were pretreated with LPS for 4 h and either left uninfected (NI) or infected with wild-type L. pneumophila (WT L.p.) or the ΔdotA mutant for 1 h at an MOI of 10. IL-1β secretion was measured by ELISA. Data are representative of four independent experiments. *, P < 0.05 in comparison to C57BL/6 BMMs (A) or uninfected BMMs (D).

FIG. 8.

Pore formation is a host response triggered in response to different Legionella species. Different species of Legionella were used to assess pore formation (A), IL-1β release (B), direct protein staining of flagellin extractions (C), and Western blotting with antiflagellin (D). (A and B) BMMs obtained from C57BL/6 mice were pretreated with LPS for 4 h and were either left uninfected (NI) or infected for 1 h at an MOI of 50 with wild-type L. pneumophila (WT L.p.), the isogenic ΔdotA mutant, a highly virulent clinical isolate of L. pneumophila (F2111), or with the indicated species of Legionella. Infected cultures were assayed for pore formation, and the supernatants of the same experiment were assayed for secretion of IL-1β by ELISA. Bacterial species were grown on BCYE agar plates for 6 days and used to extract flagellin. The proteins were separated by SDS-PAGE and stained with Coomassie blue (C) or were blotted and probed with antiflagellin antibody raised in mouse (D). Data are representative of three independent experiments. *, P < 0.05 in comparison to noninfected BMMs.