ESCRT and autophagy cooperate to repair ESX-1-dependent damage to the Mycobacterium-containing vacuole

Abstract

24Phagocytes capture invader microbes within the bactericidal phagosome. Some pathogens subvert killing 25 by damaging and escaping from this compartment. To prevent and fight bacterial escape, cells contain and 26 repair the membrane damage, or finally eliminate the cytosolic escapees. All eukaryotic cells engage highly 27 conserved mechanisms to ensure integrity of membranes in a multitude of physiological and pathological 28 situations, including the Endosomal Sorting Complex Required for Transport (ESCRT) and autophagy 29 machineries. In Dictyostelium discoideum, recruitment of the ESCRT-III protein Snf7/Chmp4/Vps32 and 30the ATPase Vps4 to sites of membrane repair relies on the ESCRT-I component Tsg101 and occurs in 31 absence of Ca 2+ . The ESX-1 dependent membrane perforations produced by the pathogen Mycobacterium 32 marinum separately engage both ESCRT and autophagy. In absence of Tsg101, M. marinum escapes earlier 33 to the cytosol, where it is restricted by xenophagy. We propose that ESCRT has an evolutionary conserved 34 function in containing intracellular pathogens in intact compartments. 35 36 37 38 39 40 41 42 Keywords 43 ESCRT, autophagy, membrane damage, membrane repair, infection, innate immunity, ESX-1, 44 Mycobacterium-containing vacuole, Dictyostelium discoideum, Mycobacterium marinum. 45 46 3 Introduction 47After phagocytic uptake, the closely related pathogenic bacteria Mycobacterium tuberculosis and M. 48 marinum reside in an altered and maturation-arrested phagosome, thereby avoiding its toxic chemical 49 environment 1 , but remaining protected from the cell-autonomous cytosolic defences 2 . This Mycobacterium-50 containing vacuole (MCV) becomes permissive for the bacilli to survive and replicate 3, 4 . However, bacteria 51 access to nutrients is limited. To circumvent this restriction, tubercular mycobacteria damage the MCV and 52 escape to the cytosol. The site of MCV rupture becomes a complex battlefield where various machineries 53 cooperate to repair membrane damage and control cytosolic bacteria. Here, we used the Dictyostelium 54 discoideum-M. marinum system to study the role of Endosomal Sorting Complex Required for Transport 55 (ESCRT) and autophagy in membrane repair during both sterile and pathogen-induced damage. We show 56 that the function of ESCRT-III in membrane repair is evolutionarily conserved, that it contributes to the 57 integrity of the MCV and plays an unrecognised role in cell-autonomous defence. We also provide evidence 58 that the ESCRT-III and autophagy pathways act in parallel to repair endomembrane compartments, but 59 differ in their ability to restrict mycobacteria growth in the cytosol of infected cells. 60 61To access the cytosol, mycobacteria make use of a crucial pathogenicity locus, the Region of 62 Difference 1 (RD1), which encodes the ESX-1 system responsible for the secretion of the membranolytic 63 peptide ESAT-6 5 . The membrane perforations produced by ESAT-6 cause MCV rupture and bacterial 64 escape to the cytosol 3, 6, 7 , a step that precede...

Localization of all four ZnT zinc transporters in Dictyostelium and impact of ZntA and ZntB knockout on bacteria killing

“…2B,D). To demonstrate that the absence of FZ-3 signal in endosomes and lysosomes is due to low pH, cells stained with FZ-3 and 10 kDa dextran were incubated with the lysosome-disrupting agent L-leucyl-L-leucine methyl ester (LLOMe) as described previously (Lopez-Jimenez et al, 2018). LLOMe creates small pores, leading to proton leakage and reneutralization of the compartments (Repnik et al, 2017).…”

ER-dependent membrane repair of mycobacteria-induced vacuole damage

“…Overall, OSBP8 is specifically recruited to MCVs starting from early infection stages, which correlates with the occurrence of MCV-damage in D. discoideum (6).…”

“…In D. discoideum, M. marinum resides in a compartment with partially lysosomal and post-lysosomal characteristics that is exposed to damage starting from early infection stages (6,16). We investigated whether mycobacterial infection leads to the formation of ER-MCV contacts.…”

“…Importantly, the host response and course of infection by M. tuberculosis and M. marinum share a high level of similarity (4) including the molecular machinery for host lipid acquisition and turnover (5). In M. marinum-infected D. discoideum, the ESCRT machinery cooperates with autophagy to repair EsxA-mediated damage at the MCV (6). The evolutionarily conserved E3-ligase TrafE mobilises various ESCRT components to damaged lysosomes and the MCV (7).…”

“…The evolutionarily conserved E3-ligase TrafE mobilises various ESCRT components to damaged lysosomes and the MCV (7). While the ESCRT components Tsg101, Chmp4/Vps32 and the AAA-ATPase Vps4 are recruited to small membrane ruptures, the autophagy machinery operates at places of extensive membrane damage (6). When ESCRT-dependent and autophagy pathways are disrupted, M. marinum escapes to the cytosol at very early infection stages, indicating that both mechanisms are needed to keep the bacteria inside the phagosome (6).…”