Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb) is a major global health problem and causes extensive cytotoxicity in patient cells and tissues. Here we define an NLRP3, caspase-1 and gasdermin D-mediated pathway to pyroptosis in human monocytes following exposure to Mtb. We demonstrate an ESX-1 mediated, contact-induced plasma membrane (PM) damage response that occurs during phagocytosis or from the cytosolic side of the PM after phagosomal rupture in Mtb infected cells. This PM injury in turn causes K+ efflux and activation of NLRP3 dependent IL-1β release and pyroptosis, facilitating the spread of Mtb to neighbouring cells. Further we reveal a dynamic interplay of pyroptosis with ESCRT-mediated PM repair. Collectively, these findings reveal a novel mechanism for pyroptosis and spread of infection acting through dual PM disturbances both during and after phagocytosis. We also highlight dual PM damage as a common mechanism utilized by other NLRP3 activators that have previously been shown to act through lysosomal damage. Graphical abstract


Introduction
Mycobacterium tuberculosis (Mtb) is a deadly human pathogen, causing about 1,6 million deaths 2 per year 1 . A pathological hallmark of Mtb infection is extensive necrosis in infected tissues 2 . 3 Necrosis has long been regarded as an unregulated type of cell death, but recently several 4 programmed necrotic pathways have been identified 3,4 . A highly inflammatory form of 5 programmed necrosis is pyroptosis, occurring mainly in myeloid cells after pattern-recognition 6 receptor (PRR) activation. In the classical pathway, activation of nucleotide-binding 7 oligomerization domain-like receptors (NLRs) or absent in myeloma 2 (AIM2)-like receptors 8 (ALRs) by pathogen-or self-ligands drives the macromolecular assembly of an inflammasome 9 consisting of oligomerized NLRs or ALRs, the adaptor apoptosis-associated speck-like protein 10 containing a CARD (ASC), and caspase-1 5-7 . Autocatalytic activation and cleavage of caspase-1 11 enables cleavage of pro-inflammatory cytokines interleukin (IL)-1β and IL-18, as well as the 12 pore-forming molecule gasdermin D (GSDMD) 8,9 . IL-1β is released through GSDMD pores and 13 in larger amounts during pyroptosis, the lytic cell death that often follows GSDMD pore 14 formation 10-13 . 15 16 Canonical NLRP3 and AIM2 inflammasome activation has been implicated in IL-1b release 17 during Mtb infection in mouse and human macrophages [14][15][16][17][18][19] . The agonist of AIM2 is double- activation for a range of triggers 28 with some exceptions involving mitochondrial disruption and 25 mtROS production, e.g. by the small-molecule compound Imiquimod 29 . Mitochondrial 26 dysfunction seems closely tied to Mtb-induced necrosis as well 33,34 , but whether this is related to 27 inflammasome activation is not clear. Inflammasome activation and pyroptosis by Mtb is 28 dependent on the type VII secretion system 6-kDa early secretory antigenic target (ESAT-6) 29 secretion system 1, ESX-1. ESX-1 secretes a range of protein substrates, and is postulated to

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Mtb H37Rv infection induces canonical NLRP3 activation followed by pyroptosis 48 Based on previously published findings [15][16][17][18][19] , we hypothesized that Mtb induces the assembly of 49 one of the ASC-dependent inflammasomes, NLRP3 or AIM2, upon infection of macrophages. To distinguish morphological changes that occurred as a consequence of pyroptosis from those 118 caused by Mtb infection prior to or during inflammasome activation, we infected cells for 5h with 119 Mtb-BFP in the presence of the caspase inhibitors Z-VAD and VX765. Cells were imaged live 120 during infection with DRAQ7 in the medium, and chemically fixed within two minutes of the last 121 captured frame. Cells with an ASC speck but without influx of DRAQ7 and no visible changes to Next, we investigated the structure of the ASC speck using the correlative imaging approach.   proportion to ΔΨm 57 . We observed that ΔΨm was stable in infected cells until ASC speck 189 formation, and that the potential quickly dropped after ASC speck formation (measured by a drop   One of the main effects of the ESX-1 secretion system is destabilization of phagosomal 204 membranes 36-38 . We therefore asked how phagosomal rupture is related to inflammasome 205 activation and pyroptosis. Galectin-3 (Gal3) binds to exposed glycosylated proteins usually 206 confined to the inner phagosomal membrane and has been used as a marker for ruptured 207 phagosomes 16,58 . We infected THP1-Gal3-mScarlet macrophages and observed that Gal3 was 208 indeed recruited to the vicinity of wild-type Mtb, but not to MtbΔRD1 (Figure 4a), and Gal3 209 recruitment was enhanced upon infection with Mtb grown in the absence of tween (Figure 4b).

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Gal3 recruitment to Mtb phagosomes occurred prior to inflammasome activation in about 80% of 211 pyroptotic cells, and a typical sequence of events is depicted in Figure 4c and Movie S9.   The ability to disrupt phagosomes seems important for inflammasome activation by Mtb, and it 233 has been reported that phagosomal acidification is a prerequisite for the membrane damaging Movies S11 and S12), suggesting that phagosomal acidification is not a prerequisite for Mtb 241 escape into the cytosol. The LysoView signal in single cells was stable prior to ASC speck   As damage to the PM from the cytosolic side by already internalized bacteria has (to our 273 knowledge) not been described before, we wished to verify that ALG-2 recruitment to Gal3-274 positive Mtb indeed occurred at the PM. TIRF microscopy is only sensitive to the region within 275 ~100 nm from the substrate, meaning it specifically detects events occurring at the PM 72 . We 276 imaged infected THP-1 macrophages with widefield and total internal reflection (TIRF) 277 microscopy simultaneously (Figure 6c and Movie S16). From widefield imaging we could see 278 Gal3 recruitment to intracellular Mtb that were out of range for TIRF, while ALG-2 recruitment 279 was observed in TIRF and WF modes when Mtb came into the range of the TIRF excitation, 280 demonstrating that ALG-2 recruitment following Gal3 also occurs at the PM. Mtb-infected THP-1 cells. We observed a consistent influx of calcium at timepoints of ALG-2 285 recruitment ( Figure 6d, e and Movie S17), which is expected considering that ALG-2 is known to 286 be recruited by calcium influx. Calcium influx was not observed at timepoints of Gal3 287 recruitment to Mtb phagosomes (Figure 6e, Figure S6a and Movie S18). We also imaged infected  To investigate the ultrastructure of Mtb-associated PM damage we performed live-cell CLEM.

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After live-cell imaging and rapid fixation, recent ALG-2 recruitment events were imaged by 297 confocal or Airyscan microscopy, and further by FIB-SEM tomography (Figure 6h, Figure S6b).   Figure S7b). Together, these results demonstrate that Mtb-induced 331 PM damage is a trigger for NLRP3 inflammasome activation in macrophages, and that PM repair 332 mechanisms negatively regulate inflammasome activation and pyroptosis caused by Mtb.

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Silica is another NLRP3 stimulus that supposedly triggers inflammasome activation by 335 destabilizing host cell phagolysosomes after uptake 24 . We therefore thought it interesting to       The precise mechanism of NLRP3 activation has evaded the community for over a decade.         The supernatant containing bacteria was diluted in RPMI with 10% A+ serum to the indicated 555 MOI assuming 1 OD600=3x10 8 bacteria/mL, and applied to cells for 45 minutes with or without   plates and imaged live using 10x objective as above. After 24 hours, cells were fixed in 2.5% 591 glutaraldehyde in 100mM PIPES for 1h, placed upside-down in 35mm glass bottom dishes and 592 cells of interest were re-imaged using 63X1.2W objective on a Leica SP8 confocal microscope.

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In the second approach THP-1 cells were seeded and infected as above on moulded polymer 594 coverslips (ibidi) mounted in 8 well sticky-Slides (ibidi). Samples were imaged using 40X1.3oil 595 objective on a Leica SP8 confocal microscope and fixated by adding 8% paraformaldehyde 596 (PFA) and 0.2% GA in 200mM PIPES directly into imaging media at a 1:1 ratio. After 20 597 minutes fixation media was changed to fresh 4% PFA and 0.1% GA in 100mM PIPES. Cells with 598 interesting events during live microscopy were then re-imaged with a 40X1.2Imm AutoCorr 599 objective on a Zeiss LSM880 microscope using the Airyscan mode. After re-imaging fixation 600 medium was changed to 2% PFA and 2.5% GA in 100mM PIPES and fixated at 4°C over night.     The data that support the findings of this study and the custom scripts and pipelines for image 658 analysis are available from the Lead Contact upon reasonable request.                                            (a) THP1 ASC-GFP cells were infected with different MOI of MtbH37Rv. Draq7+ cells, ASC specks and IL-1b release was measured after 24h. Data representative of 2-3 independent experiments. (b) THP1 ASC-mNeonGreen cells and THP1 ASC-mNeonGreen depleted of NLRP3 or GSDMD by CRISPR-Cas9 were lysed and protein levels measured by western blotting. (c) THP1 ASC-GFP cells were stimulated by LPS (10ng/mL, 3h), not treated or treated with DMSO, Z-VAD-FMK (50µM), VX765 (50µM), MCC950 (10µM) or KCl (40mM) and nigericin (10µM, 1h). Draq7+ cells and ASC specks were quantified from n>2000 cells per condition in triplicate, and IL-1b in the supernatant was quantified by ELISA. (d) THP1 ASC-mNeonGreen cells and THP1 ASC-mNeonGreen depleted of NLRP3 by CRISPR-Cas9 were stimulated by LPS and nigericin as above, and Draq7+ cells and ASC specks were quantified from n>2000 cells in triplicates. (e) THP1 ASC-GFP cells were treated by DMSO, GSK'872 (5µM) or Nec1s (10µM) and infected by Mtb H37Rv (MOI 20). Draq7+ cells and ASC specks were quantified 24h p.i. for n>2000 cells per condition in triplicate. (f) THP1 ASC-GFP cells were untreated or stimulated with FSL-1, Pam3Cys4K or LPS (all 10ng/mL) for 3h or 24h prior to infection with MtbH37Rv and analysed 24h p.i. for Draq7+ cells and ASC specks. n>2000 cells per condition in triplicate were quantified. Single slices from FIB-SEM tomography of (a) THP1 ASC-GFP cell treated with LPS (10ng/mL, 3h) and nigericin (10µM, 1h) (b) THP1 ASC-mNeonGreen depleted of GSDMD by CRISPR-Cas9, infected with Mtb H37Rv and xed after ASC speck formation while the cell was still alive (DRAQ7 negative and normal morphology). ASC specks indicated by arrows.