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.

In contrast to mechanisms mediating uptake of intracellular bacterial pathogens, bacterial egress and cell-to-cell transmission are poorly understood. Previously, we showed that the transmission of pathogenic mycobacteria between phagocytic cells also depends on nonlytic ejection through an F-actin based structure, called the ejectosome. How the host cell maintains integrity of its plasma membrane during the ejection process was unknown. Here, we reveal an unexpected function for the autophagic machinery in nonlytic spreading of bacteria. We show that ejecting mycobacteria are escorted by a distinct polar autophagocytic vacuole. If autophagy is impaired, cell-to-cell transmission is inhibited, the host plasma membrane becomes compromised and the host cells die. These findings highlight a previously unidentified, highly ordered interaction between bacteria and the autophagic pathway and might represent the ancient way to ensure nonlytic egress of bacteria.autophagy | Dictyostelium discoideum | Mycobacterium marinum | ejection

Cytosolic Mycobacterium marinum are ejected from host cells such as macrophages or the amoeba Dictyostelium discoideum in a non‐lytic fashion. As described previously, the autophagic machinery is recruited to ejecting bacteria and supports host cell integrity during egress. Here, we show that the ESCRT machinery is also recruited to ejecting bacteria, partially dependent on an intact autophagic pathway. As such, the AAA‐ATPase Vps4 shows a distinct localization at the ejectosome structure in comparison to fluorescently tagged Vps32, Tsg101 and Alix. Along the bacterium engaged in ejection, ESCRT and the autophagic component Atg8 show partial colocalization. We hypothesize that both, the ESCRT and autophagic machinery localize to the bacterium as part of a membrane damage response, as well as part of a “frustrated autophagosome" that is unable to engulf the ejecting bacterium.

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...

The recombinant antibodies RB155, RB156 and RB189 detect by ELISA a fragment of the D. discoideum Tsg101 fused to a GST protein.