ER-dependent membrane repair of mycobacteria-induced vacuole damage

Abstract

Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis, the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the Dictyostelium discoideum/Mycobacterium marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as M. tuberculosis.

Keywords: Dictyostelium discoideum; Mycobacterium marinum; Mycobacterium tuberculosis; Sac1; lysosome; macrophages; membrane contact site; membrane repair; oxysterol-binding protein; phosphatidylinositol 4-phosphate; sterol.

Fig 1

Evidence for ER-mediated repair and mobilization of OSBP8 during mycobacterial infection. (A and B) Proteomics (left) and heatmaps (right) represent the transcriptional data derived from references and . Cells were infected with GFP-expressing M. marinum wild type (wt). Samples were collected at the indicated time points. Statistically significant differences in expression are marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Colors indicate the amplitude of expression (in logarithmic fold change [Log2FC]) in infected cells compared to mock-infected cells: from red (highest expression) to blue (lowest expression). (C and D) Correlative light and electron microscopy (CLEM) to monitor ER-tubules close to ruptured MCVs. Cells expressing GFP-ABD and AmtA-mCherry were infected with eBFP-expressing M. marinum. At 24 h post infection (hpi), cells on sapphire discs were imaged by SD microscopy in the presence of low concentrations of GA before high-pressure freezing. Left: deconvolved SD images, scale bars, 5 µm; right: representative EM micrographs, scale bars, 500 nm. Magenta arrow heads point to the ruptured MCV membrane. Mitochondria (Mit) were indicated in orange text, M. marinum (M.m.) were pseudo-colored in cyan and ER-tubules in yellow. (E) Live cell imaging of OSBP8-GFP during infection. Cells overexpressing OSBP8-GFP were infected with mCherry-expressing M. marinum. At the indicated time points, cells were imaged live by SD microscopy. Arrows point to OSBP8-GFP⁺ mycobacteria. Scale bars, 5 µm; Zoom, 2 µm. Images in (C and E) were deconvolved.

Fig 2

Subcellular localization of OSBP8-GFP during infection. (A) LLSM to monitor OSBP8-GFP close to the MCV. (B) Intensity profile of the line plotted through the MCV shown in the zoom of panel A. (C) 3D model of the cell shown in panel A illustrating OSBP8-GFP⁺ membranes capping the MCV (AmtA⁺). Cells dually expressing OSBP8-GFP/AmtA-mCherry were infected with eBFP-expressing M. marinum and imaged live at 3 hpi by LLSM. Arrow points to OSBP8-GFP⁺ membranes close to the MCV. Scale bars in A, 5 µm; Zoom, 2 µm; in C, 2 µm. (D) Expansion microscopy to visualize ER-MCV MCS during infection. Cells dually expressing OSBP8-GFP/Calnexin-mCherry were infected with eBFP-expressing M. marinum, fixed at 24 hpi, and stained with antibodies against p80, GFP, and mCherry before 4× expansion. Arrow points to an OSBP8-GFP⁺ ER-tubule close to the MCV. Scale bar, 20 µm; Zoom, 1 µm. Images were deconvolved. (E) 3D-CLEM to visualize OSBP8-mCherry⁺ ER-tubules close to the MCV (arrow). Cells expressing OSBP8-mCherry/GFP-ABD were infected with eBFP-expressing M. marinum. At 24 hpi, cells were imaged by SD microscopy (E) and prepared for SBF-SEM (F and G). (F) EM micrograph showing the cell with the correlated OSBP8-mCherry and eBFP-M. marinum signal. Please see Fig. S3C for more information. (G) Closeup of the position indicated in (E and F) showing ER-tubules close to the MCV. (i–iv) correlation of the (i) EM micrograph (ii) with OSBP8-mCherry (magenta) and mycobacteria (blue), (iii and iv) segmentation of the ER (yellow), MCV (violet), and mycobacteria (cyan). Scale bars, 5 µm (E); 2 µm (F); and 1 µm (G). SD images were deconvolved. N, nucleus.

Fig 3

OSBP8-GFP localization during infection with mycobacterial mutants and cells additionally expressing P4C-GFP. (A) Localization of OSBP8-GFP during infection with the M. marinum ΔRD1 mutant. (B) Quantification of panel A. Data represent two independent experiments (OSBP8-GFP 3, 21, 27, 46 hpi N = 2, 23 ≤ n ≤ 274). (C) Localization of OSBP8-GFP during infection with the M. marinum ΔCE mutant. (D) OSBP8-GFP localization during infection in cells highly expressing P4C-mCherry. Cells overexpressing OSBP8-GFP or co-expressing P4C-mCherry were infected with mCherry- or eBFP-expressing M. marinum wt, ΔRD1, or ΔCE. At the indicated time points, samples were taken for SD microscopy. Arrows point to OSBP8-GFP^- intracellular mycobacteria. Arrowheads indicate PI4P⁺ MCV. Scale bars, 5 µm; Zoom, 2.5 µm. Images were deconvolved. M.m., M. marinum.

Fig 4

Effect of OSBP8 deletion on PI4P distribution, lysosomal and degradative properties of MCVs, and mycobacterial growth. (A) P4C-GFP distribution on MCVs in the osbH KO. (B) Quantification of panel A. Plots show the mean and standard deviation of one of two independent experiments (P4C-GFP 3, 27 hpi N = 2, 14 ≤ n ≤ 24). Statistical differences were calculated with an unpaired t-test (**P < 0.01). (C) Sterol distribution in wt vs osbH KO cells infected with M. marinum wt. (D) Quantification of panel C. Plots show the mean and standard deviation of three independent experiments (Filipin 8, 21 hpi N = 3, 30 ≤ n ≤ 70). Statistical differences were calculated with an unpaired t-test (****P < 0.0001). (E) Lysosomal properties of MCVs in wt vs osbH KO cells. (F and G) Quantifications of panel E. Plots show the mean and standard deviation of three independent experiments (LysoSensor green 3, 27 hpi N = 3, 290 ≤ n ≤ 450). Statistical differences were calculated with an unpaired t-test (*P < 0.05; **P < 0.01; ****P < 0.0001). (H) Proteolytic activity of MCVs in wt vs osbH KO cells. (I and J) Quantification of panel H. Plots show the mean and standard deviation of three independent experiments (DQ-BSA green 3, 27 hpi N = 3, 140 ≤ n ≤ 240). Statistical differences were calculated with an unpaired t-test (****P < 0.0001). D. discoideum wt and osbH KO (expressing P4C-GFP [A]) were infected with mCherry- or eBFP-expressing M. marinum wt. At the indicated time points, samples were taken for SD microscopy. For filipin staining, infected cells were fixed and stained for VatA. Arrows point to PI4P⁺ or LysoSensor⁺ or DQ-BSA⁺ MCVs and arrow heads indicate sterol accumulation inside MCVs. Scale bars, 5 µm. Images in panel C were deconvolved. M.m., M. marinum. (K and L) Mycobacterial growth in cells lacking OSBP8 or cells overexpressing OSBP8-GFP. D. discoideum wt, two independent osbH KOs or OSBP8-GFP overexpressing cells were infected with M. marinum wt expressing bacterial luciferase. Luminescence was recorded every hour with a microplate reader. Shown is the fold increase in luminescence over time. Symbols and error bands indicate the mean and standard error of three independent experiments. Statistical differences were calculated with a Tukey post hoc test after two-way ANOVA (****P < 0.0001).

Fig 5

Evidence for ER-mediated repair and OSBP mobilization during M. tuberculosis infection. (A and B) Heatmaps of differentially expressed genes (Log2FC) encoding proteins involved in MCS formation from RNA-sequencing analysis of human iPSDMs infected with either M. tuberculosis wt or ΔRD1. Data were retrieved from reference (13). Samples were collected at the indicated time points. Statistically significant differences in expression are marked with asterisk (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Colors indicate the amplitude of expression (Log2FC) in infected cells compared to mock-infected cells: from red (highest expression) to blue (lowest expression). (C and D) Localization of endogenous OSBP in human iPSDMs upon infection with M. tuberculosis wt and the ΔRD1 mutant. iPSDMs were infected with E2-Crimson-expressing wt or ΔRD1 bacteria. At 2 and 48 hpi, cells were fixed and stained against OSBP. Shown are two representative images from 48 hpi. Z-stacks: 20, 0.3 µm. Arrows and arrowheads point to OSBP8⁺ or OSBP8⁻ intracellular bacteria, respectively. Scale bars, 5 µm; Zoom; 2 µm. (E) Quantification of (C and D). Plots show the mean and standard deviation of three independent experiments (OSBP 2, 48 hpi N = 3, 800 ≤ n ≤ 1,200). Statistical differences were calculated with an unpaired t-test (****P < 0.0001). Mtb, M. tuberculosis.

Fig 6

Schematic outline of ER-dependent repair during mycobacterial infection. (1) ESX1-dependent vacuolar damage (yellow flash) leads to a loss of ion gradients (green spots) and the release of proteases (green packmen). (2) PI4K is recruited to generate PI4P (pink polygons) at the MCV. (3) This leads to the establishment of ER-MCV MCS and the mobilization of OSBP8 (blue) in M. marinum-infected D. discoideum. (4) OSBP8 transports sterols from the ER to the MCV and PI4P in the opposite direction. (5) The transport is fueled by the lipid phosphatase Sac1 that hydrolyses PI4P-generating PI (light pink).