Host-detrimental role of Esx-1-mediated inflammasome activation in mycobacterial infection

Abstract

The Esx-1 (type VII) secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium marinum. However, the molecular events and host-pathogen interactions underlying Esx-1-mediated virulence in vivo remain unclear. Here we address this problem in a non-lethal mouse model of M. marinum infection that allows detailed quantitative analysis of disease progression. M. marinum established local infection in mouse tails, with Esx-1-dependent formation of caseating granulomas similar to those formed in human tuberculosis, and bone deterioration reminiscent of skeletal tuberculosis. Analysis of tails infected with wild type or Esx-1-deficient bacteria showed that Esx-1 enhanced generation of proinflammatory cytokines, including the secreted form of IL-1beta, suggesting that Esx-1 promotes inflammasome activation in vivo. In vitro experiments indicated that Esx-1-dependent inflammasome activation required the host NLRP3 and ASC proteins. Infection of wild type and ASC-deficient mice demonstrated that Esx-1-dependent inflammasome activation exacerbated disease without restricting bacterial growth, indicating a host-detrimental role of this inflammatory pathway in mycobacterial infection. These findings define an immunoregulatory role for Esx-1 in a specific host-pathogen interaction in vivo, and indicate that the Esx-1 secretion system promotes disease and inflammation through its ability to activate the inflammasome.

Figure 1. Disease and inflammation can be quantified and indicate a major role for Esx-1 in virulence.

(A) B6 mice were infected via tail vein injection with either wild type or ΔRD1 bacteria, and observed for appearance of visible tail lesions. Shown are representative tails at indicated times post infection. (B) Quantification of the accumulated length of all visible lesions in individual tails at indicated times. Solid lines indicate the mean for each group. (C) Micro-CT scans of tails infected with wild type or ΔRD1 bacteria. Uninfected control mice were injected with PBS. Shown are 3D-renderings for one representative tail from each group at indicated times post infection. (D) Mean bone volume at indicated times. Values represent mean ± SD of three mice per group. Student's t-test (*P<0.05; **P<0.01).

Figure 2. M. marinum cause formation of granulomas, similar to those formed in human tuberculosis, in an Esx-1-dependent manner.

B6 mouse tails infected with wild type or ΔRD1 M. marinum were analyzed for formation of granulomatous lesions by hematoxylin and eosin (H&E), as well as anti-CD3 (T cells) and anti-F4/80 (macrophages) immuno histochemistry. Immunostained cells appear brown. Lesion borders are indicated with a red dotted line for clarity. (A) 14 days post infection. (B) 21 days post infection. (C) 28 days post infection. (D) Top panel: H&E staining of a typical granuloma in wild type infected tails 21 days post infection. Center contains acellular necrosis (indicated with an asterix). Lower panel: Higher magnification of region with acellular necrosis (representative areas are indicated with arrows).

Figure 3. Esx-1 promotes bacterial growth and phagosome escape in tails of infected mice.

(A) Bacterial burdens in the blood of wild type and ΔRD1 infected B6 mice at indicated time points. Dotted line indicates detection level. (B) Bacterial burden in lung, liver and tail tissues in wild type and ΔRD1 infected mice, as indicated. Values are mean + SD of three mice per group (A and B). The statistical difference between wild type and ΔRD1 infected tails was calculated with Student's t-test (*P<0.05; ***P<0.001). Similar statistical analysis indicated a significant (P<0.05) bacterial growth in wild type infected tails between day 1 and 21. The mean CFU/g tail tissue during the first 21 days in wild type infected mice were as follows: day 1: 1.8×10⁷; day 14: 5.82×10⁷; day 21: 9.95×10⁷. (C) TEM analysis of infected cells in lesions in wild type (left and middle panels) and ΔRD1 (right panel) infected tails. Upper panels: Cell morphology, degree of nuclear condensation, amount of cytoplasm and cytoplasmic granules suggested infected cells were macrophages. Cell membranes are indicated with red lines, nuclei with n, and areas with intracellular bacteria with white boxes. Lower panels: Higher magnification of areas with intracellular bacteria. Intraphagosomal bacteria are indicated with an encircled asterix, and cytosolic bacteria with an asterix. Red arrows point to membranes of bacteria-containing vesicles. Question mark indicates a bacterium with undeterminable localization. (D) Quantification of cytosolic and intraphagosomal wild type (n = 142) and ΔRD1 (n = 389) bacilli in infected tails by TEM. ‘Undeterminable’ indicates bacteria with uncertain localization.

Figure 4. Esx-1 promotes secretion of IL-1β in vivo.

(A) 20 days post infection tail suspensions were prepared from uninfected controls, wild type and ΔRD1 infected mice, and analyzed by Luminex for indicated cytokines. Data are presented as the amount of cytokine detected (pg/ml) divided by the total protein content of the suspensions (mg/ml). Values are mean ± SD of three mice per group. (B) Tail suspensions from three mice per group were separated by SDS-PAGE, and analyzed for mature IL-1β and actin by immunoblot. For each tail, the amount of mature IL-1β was divided by the amount of actin, and normalized to the wild type infected mouse with the highest ratio. Values are mean ± SD for each group. Student's t-test (*P<0.05; **P<0.01).

Figure 5. Esx-1 is required for activation of the NLRP3/ASC-inflammasome in bone marrow-derived macrophages.

(A) B6 macrophages were infected with wild type M. marinum for 4, 8 and 12 hrs, as indicated, and analyzed for caspase-1 activation by anti-caspase-1 p10 immunoblot. Uninfected macrophages were analyzed as control. (B) Macrophages were infected as indicated, and analyzed for caspase-1 p10 12 hrs post infection. (C) B6 macrophages were infected as indicated, and analyzed for caspase-1 p10 12 hrs post infection. (D) Macrophages were infected (MOI = 0.1) as indicated, and bacterial growth determined by CFU-analysis. (E) Macrophages were infected with wild type (left panel) or ΔRD1 (right panel) and analyzed for cell death. Solid lines represents infected cells, and dotted lines represents uninfected controls. At indicated time points, supernatants were analyzed for LDH-release as a measure of loss of host cell membrane integrity (i.e. host cell death). Data are presented as relative LDH-release; 100% LDH-release was defined by lysis of uninfected cells with Triton-X100 treatment. Shown are representative data for at least three separate experiments (A to E).

Figure 6. Activation of the inflammasome exacerbates inflammation without restricting bacterial growth in vivo.

(A) Mice were infected as indicated, and analyzed for accumulated length of all visible tail lesions. Lines indicate the mean for each group (n = 11 per group). (B) Mean ± SD bone volume determined by micro-CT of three mouse tails for each group at 21 days post infection. Statistical significance of differences between B6 and ASC-KO mice infected with wild type M. marinum was calculated by Student's t-test (*P<0.05; **P<0.01) (A and B). (C) 21 days post infection; tails of B6 and ASC-KO mice were analyzed for granuloma formation by H&E as well as anti-CD3 and anti-F4/80 immunohistochemistry. Lesion borders are indicated with a red dotted line for clarity. (D) Bacterial burdens in tails of infected mice, as indicated, at 21 days post infection. Values are mean CFU ± SD of three mice per group.