The ESCRT and autophagy machineries cooperate to repair ESX-1-dependent damage at the Mycobacterium-containing vacuole but have opposite impact on containing the infection

Abstract

Phagocytic cells capture and kill most invader microbes within the bactericidal phagosome, but some pathogens subvert killing by damaging the compartment and escaping to the cytosol. To prevent the leakage of pathogen virulence and host defence factors, as well as bacteria escape, host cells have to contain and repair the membrane damage, or finally eliminate the cytosolic bacteria. All eukaryotic cells engage various repair mechanisms to ensure plasma membrane integrity and proper compartmentalization of organelles, including the Endosomal Sorting Complex Required for Transport (ESCRT) and autophagy machineries. We show that during infection of Dictyostelium discoideum with Mycobacterium marinum, the ESCRT-I component Tsg101, the ESCRT-III protein Snf7/Chmp4/Vps32 and the AAA-ATPase Vps4 are recruited to sites of damage at the Mycobacterium-containing vacuole. Interestingly, damage separately recruits the ESCRT and the autophagy machineries. In addition, the recruitment of Vps32 and Vps4 to repair sterile membrane damage depends on Tsg101 but appears independent of Ca2+. Finally, in absence of Tsg101, M. marinum accesses prematurely the cytosol, where the autophagy machinery restricts its growth. We propose that ESCRT has an evolutionary conserved function to repair small membrane damage and to contain intracellular pathogens in intact compartments.

Fig 1. The ultrastructure of the MCV at the point of rupture reveals a complex battlefield between the M. marinum and its host.

D. discoideum was infected with M. marinum and fixed at 24 hpi for visualization by FIB-SEM. (A) Sections of the same infected cell, showing a disrupted MCV (blue), M. marinum (red) accessing the cytosol and a potential autophagosome (green) engulfing the pole of a cytosolic bacterium. Green arrowheads point to a complex structure with discontinuities and electron-dense boundaries surrounding the cytosolic M. marinum. Squares delimitate the areas of interest magnified in C. (B) 3D reconstruction of the FIB-SEM stack shown in A. (C) The cytosolic bacteria were surrounded by a structure (green arrowheads) with very electron-dense boundaries (pink asterisks). The cytosolic material between the bacteria and this structure or the autophagosome was slightly more electron-dense than the rest of the cytosol (blue asterisks) (D). Section of a cell, showing a disrupted MCV (blue), M. marinum (red) and the dark electron-dense material surrounding the sites of escape (blue asterisks, yellow). (E) 3D reconstruction of the FIB-SEM stack shown in D (see also S1 Movie). Abbreviations: M. m., M. marinum; MCV, Mycobacterium-containing vacuole; Mit, Mitochondria; Atg, Autophagosome. Scale bars, 1 μm.

Fig 2. Recruitment of ESCRT components to the MCV upon M. marinum induced damage.

(A) D. discoideum expressing GFP-Tsg101, GFP-Vps32 or Vps4-GFP were infected with M. marinum wt or M. marinum ΔRD1, and z-stacks were acquired at 1.5, 5, 8, 24 and 31 hpi. Maximum projections are shown. GFP-Tsg101, GFP-Vps32 and Vps4-GFP structures (white arrows) appeared in the vicinity of the MCV containing M. marinum wt (red), and to a lesser extent around M. marinum ΔRD1 (red). Scale bars, 5 μm. (B-D) Quantification of GFP-Tsg101 structures (patches and foci) and GFP-Vps32 and Vps4-GFP structures (patches and rings) in the vicinity of M. marinum wt or M. marinum ΔRD1. Plots show the mean and standard deviation (GFP-Tsg101 1.5, 8, 24, 31 hpi N = 3, 38≤n≤198; 5 hpi N = 2, 59≤n≤117, GFP-Vps32 N = 3 32≤n≤145; Vps4-GFP N = 3, 64≤n≤233). (E) D. discoideum expressing GFP-Vps32 were infected with M. marinum wt (red) and monitored by time-lapse microscopy every 3 min (see also S2 Movie). Maximum projections of the same cell are shown (time indicated in the top right corner). GFP-Vps32 rings formed and appeared to move along the bacterium (arrows). Bottom panels show insets focused on the ring structures (arrows). Scale bars, 10 μm and 1 μm for the insets. (F) Section of a z-stack showing the recruitment of GFP-Vps32 to the vicinity of the MCV (bacteria in red) at 31 hpi. Projections of the xz and yz planes are shown. Scale bar, 10 μm. (G) D. discoideum wt or tsg101- expressing GFP-Vps32 were infected with M. marinum (in red) and z-stacks were acquired at 1.5, 8 and 24 hpi. Maximum projections are shown. GFP-Vps32 was recruited to a lesser extent to the MCV in tsg101- cells. Scale bars, 5 μm. (H) Quantification of GFP-Vps32 structures formed in the vicinity of M. marinum in wt or tsg101- cells. The plot shows the mean and standard deviation (N = 3, 76≤n≤154). Two-way ANOVA and post hoc Fisher’s LSD tests were performed.

Fig 3. GFP-Vps32 and GFP-Atg8 are recruited to the disrupted MCV at spatially distinct sites.

(A) D. discoideum expressing GFP-Vps32 were infected with M. marinum (blue) and fixed for immunostaining against p80 to label the MCV (red). At 1.5 hpi, no GFP-Vps32 structure was visible at apparent intact MCVs. At 8 and 24 hpi, GFP-Vps32 foci and patches appeared in the vicinity of the bacteria, where the MCV was evidently disrupted (arrows). Scale bar, 10 μm and 1 μm for the insets. (B-C) D. discoideum expressing GFP-Vps32 and AmtA-mCherry were infected with M. marinum (blue) and monitored by time-lapse microscopy. (B) Still images show GFP-Vps32 patches on the disrupted MCV (arrows) (see also S2A–S2C Fig). (C) Time-lapse showing the dynamics of association of GFP-Vps32 to the AmtA-mCherry labelled MCV (see also S3 Movie). Scale bars, 10 μm and 1 μm for the insets. (D) D. discoideum was infected with M. marinum and fixed for immunostaining at 24 hpi. Only bacteria (blue) that have escaped the MCV (p80, red) showed ubiquitin structures (green). Scale bar, 5 μm. (E-F) D. discoideum expressing GFP-Vps32 were infected with M. marinum (blue) and fixed for immunostaining at 8 and 24 hpi to visualize ubiquitin or Atg8 (red). GFP-Vps32 and ubiquitin or Atg8 were recruited to the same macroscopic region of the MCV, but they did not perfectly colocalise. GFP-Vps32 formed patches devoid of ubiquitin or Atg8 staining (white arrows). Vice versa, ubiquitin and Atg8 appeared in areas where no GFP-Vps32 was observed (yellow arrows). Scale bar, 5 μm and 1μm for the insets. (G) Schematic representation of the Vps32, ubiquitin and Atg8 recruitments at the damaged MCV.

Fig 4. Differential spatial and temporal recruitment of ESCRT components and Atg8 upon sterile damage.

D. discoideum expressing GFP-Tsg101, GFP-Vps32, Vps4-GFP or GFP-Atg8 were subjected to membrane disrupting agents and monitored by time-lapse imaging. (A) Treatment with digitonin led to the appearance of the three ESCRT-components but not GFP-Atg8 at the plasma membrane within minutes (see also S4 Movie). (B) Cells were incubated with 10 kDa fluorescent dextran for at least 3 h to label all endosomal compartments and treated with LLOMe (see also S5 Movie). Punctate ESCRT-structures appeared at the periphery of the lysosomes as early as 2.5 min after the addition of LLOMe. More diffuse, ring-like GFP-Atg8 structures were visible about 5 min later. Scale bars, 10 μm or 1 μm for the insets. (C-D) Quantification of the time-lapse experiments shown in (A-B), respectively. The proportion of the cell area occupied by the structures in maximum projections is plotted as a function of time. Graphs show the mean and SEM of N≥3 independent experiments.

Fig 5. Mechanistic characterization of ESCRT-III and autophagy recruitment and repair of membrane damage.

(A) Schematic representation of the experiment shown in B. Annexin V binds to PS exposed upon plasma membrane disruption. (B) D. discoideum were incubated with Annexin V Alexa Fluor 594 conjugate in the presence of Ca²⁺ and then treated with digitonin and monitored by time-lapse imaging (see also S5 Movie). GFP-Vps32 structures (in green) appeared in close proximity of Annexin V-positive structures (in red, white arrows). At later times, the Annexin V-positive structure was released into the medium (yellow arrows), leaving a GFP-Vps32 “scar” (white arrow). Time is indicated in the bottom left corner. (C-D) In cells lacking Tsg101, neither digitonin nor LLOMe treatment led to the formation of GFP-Vps32 structures. (D) Endosomes (in red) were labelled with 10 kDa fluorescent dextran for at least 3 h. (E) Cells were incubated with EGTA or mock-incubated, treated with digitonin and monitored by time-lapse microscopy. Neither spatial nor temporal differences in GFP-Vps32 recruitment (white arrows) were observed. (F) Cells were incubated with EGTA and BAPTA-AM or mock-incubated, treated with LLOMe and monitored by time-lapse microscopy. Neither spatial nor temporal differences in GFP-Vps32 recruitment (white arrows) were observed. Scale bars correspond to 10 μm. (G-J) Quantification of the time-lapse experiments shown in (C-F), respectively. The proportion of the cell area occupied by the structures in maximum projections is plotted as a function of time. Graphs show the mean and SEM of N = 3 independent experiments.

Fig 6. Increased leakage of lysosomes in ESCRT and autophagy mutants upon LLOMe treatment.

(A) Schematic representation of the experiment shown in B. Cells were treated for at least 3 h with the Alexa Fluor 647 10 kDa dextran (red) to label all endosomes, together with the 0.5 KDa soluble pH indicator HPTS (green). (B) D. discoideum wt, tsg101- and atg1- were subjected to the experimental procedure depicted in A and monitored by time-lapse imaging (see also S7 Movie). Before addition of LLOMe, HPTS was quenched in acidic lysosomes, which therefore appeared in red. HPTS dequenching started after 30 sec in tsg101- and atg1- cells and after 1.5 min in wt cells. (C) Quantification of the time-lapse experiments shown in B. The ratio of the intensities of HPTS and Alexa Fluor 647 10 kDa dextran in sum projections is plotted as a function of time. The graph shows the mean and SEM of N = 10 independent experiments. Two-way ANOVA and post hoc Fisher’s LSD tests were performed.

Fig 7. Deficiency in membrane repair leads to earlier escape of M. marinum from the MCV.

(A) D. discoideum wt or mutant (atg1-, tsg101-, atg1- tsg101-) were infected with M. marinum and fixed for immunostaining at 8 hpi (M. marinum in red, ubiquitin in green, DAPI in blue). Arrows point to ubiquitinated bacteria. Scale bars, 10 μm and 5 μm for the insets. (B) Quantification of the proportion of ubiquitinated bacteria or bacterial microcolonies. The plot shows the mean and standard deviation (WT N = 6, n = 306; atg1- N = 3, n = 251; tsg101- N = 3, n = 93; atg1- tsg101- N = 3, n = 215). (C) Wt or tsg101- cells were infected with M. marinum and fixed for immunostaining at 8 hpi (M. marinum in red, Atg8 in green, DAPI in blue). Arrows point to bacteria decorated with Atg8. Scale bars, 10 μm and 5 μm for the insets. (D) Quantification of the proportion of bacteria or bacterial microcolonies decorated with Atg8. The plot shows the mean and standard deviation. (WT N = 3, n = 70; tsg101- N = 3, n = 63). Two-tailed t-tests (D) or two-way ANOVA and post hoc Fisher’s LSD tests were performed (B).

Fig 8. Impact of ESCRT and autophagy on mycobacterial growth.

(A-B) Wt or mutant D. discoideum were infected with luminescent M. marinum and intracellular bacterial growth was monitored in a plate reader over 72 hpi. M. marinum growth was restricted in the tsg101- mutant, whereas it hyperproliferates in the atg1- and the double atg1- tsg101- mutant. Plots represent the mean and standard deviation of N = 3 independent experiments. Two-way ANOVA and post hoc Fisher’s LSD test were performed. (C) Model of autophagy and ESCRT-III involvement in MCV repair and restriction of M. marinum intracellular growth. In wt D. discoideum, M. marinum induces ESX-1-dependent injuries in the MCV membrane. This leads to the separate recruitment of the ESCRT-III and autophagy machineries to repair the damage, but nevertheless the bacteria are able to access the cytosol at later stages of infection (24–48 hpi). In the atg1- mutant, M. marinum access the cytosol earlier, despite the membrane repair exerted by ESCRT-III. Bacteria accumulate ubiquitin and proliferate more extensively in the cytosol devoid of a functional autophagy pathway. In the tsg101- mutant, membrane damage not repaired by ESCRT-III leads to an increase of ubiquitination and recruitment of the autophagy machinery, resulting in restriction of M. marinum growth. In a double atg1- tsg101- mutant, lack of both ESCRT-III and autophagic membrane repairs leads to an earlier access of bacteria to the cytosol, which accumulate ubiquitin and hyperproliferate as in the single atg1- mutant.