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.

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

“…Since PM damage is actively repaired through many routes, including through Ca2+ - dependent ESCRT machinery 44, 69–71 , it is also interesting to note that this branch of ESCRT emerges as a negative regulator of NLRP3 activation and cell death during Mtb infection. The ESCRT machinery was also recently shown to be involved in repair of phagosomes damaged by mycobacteria 84, 85 and to regulate pyroptosis or necroptosis by repairing GSDMD pores 86 or MLKL pores 87 , respectively, further pointing to the importance of the ESCRT machinery in regulation of inflammation and securing cell viability.…”

Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes

“…marinum is widely used as a versatile M. tuberculosis infection model (Arafah et al, 2013;Bouz & Al Hasawi, 2018;Cardenal-Munoz, Barisch, Lefrancois, Lopez-Jimenez, & Soldati, 2017;Hagedorn, Rohde, Russell, & Soldati, 2009;Hagedorn & Soldati, 2007;Lienard & Carlsson, 2017;Prouty, Correa, Barker, Jagadeeswaran, & Klose, 2003;Shiloh & Champion, 2010;Tükenmez et al, 2019). M. marinum can be handled at biosafety Level 2 and is appreciated for its relatively rapid growth (doubling time of 4-10 hr vs. >20 hr for M. tuberculosis; Clark & Shepard, 1963), the M. tuberculosis-like behaviour during intracellular infection, such as replication within a distinct Mycobacterium-containing vacuole (MCV; Barker, George, Falkow, & Small, 1997;Pozos & Ramakrishnan, 2004;Smith et al, 2008;Tobin & Ramakrishnan, 2008;López-Jiménez et al, 2018;Koliwer-Brandl et al, 2019), as well as its wide spectrum of host cells including macrophages (Barker et al, 1997;Bouley, Ghori, Mercer, Falkow, & Ramakrishnan, 2001;Koliwer-Brandl et al, 2019;Ramakrishnan, Federspiel, & Falkow, 2000) and the amoeba Dictyostelium discoideum (Cardenal-Munoz et al, 2017;Hagedorn et al, 2009;Koliwer-Brandl et al, 2019;Solomon, Leung, & Isberg, 2003) or Acanthamoeba castellanii (Harrison et al, 2013;Kicka et al, 2014).…”

Zn2+ intoxication of Mycobacterium marinum during Dictyostelium discoideum infection is counteracted by induction of the pathogen Zn2+ exporter CtpC

“…On the contrary, visibly broken compartments appeared devoid of Zn 2+ , suggesting leakage due to mycobacteria-induced perforation of the MCV membranes. In fact, quantification of NBD-TPEAlabelled MCVs confirmed that, whilst wild-type (wt) MCVs were less positive for Zn 2+ with progression of infection, intact MCVs containing M. marinum ∆RD1, a mutant lacking the ESX-1 secretion system and thus strongly attenuated in its capacity to induce membrane damage (Hagedorn et al, 2009;Lopez-Jimenez et al, 2018), remained positive for Zn 2+ during the whole infection cycle (Fig. 1C).…”

“…M. marinum starts damaging the MCV in the first hours of infection, before full rupture of the compartment releases the bacteria into the host cytosol at around 24-36 hours. Perforation of the MCV is achieved by secretion of the membrane-damaging peptide ESAT-6 through the mycobacterial ESX-1 Type VII Secretion System (T7SS) (Cardenal-Munoz et al, 2017a;Hagedorn et al, 2009;Lopez-Jimenez et al, 2018;Smith et al, 2008). The role of ESX-1-mediated secretion in MCV-to-cytosol translocation has also been shown for Mtb (Houben et al, 2012;Mittal et al, 2018;Simeone et al, 2012;van der Wel et al, 2007).…”