Survival of intracellular pathogens in response to mTORC1- or TRPML1-TFEB-induced xenophagy.

Abstract

Intracellular pathogens establish persistent infections by generating reservoirs that protect them from the action of antibiotics and the host immune response. Novel therapeutics should then target the host pathways exploited by the pathogens to form these intracellular niches. An attractive strategy to achieve this is inducing xenophagy, the selective autophagy that recognizes and targets invading pathogens for degradation. However, some bacteria have evolved mechanisms to co-opt xenophagy for their own benefit. Therefore, in this study we determine the effect of inducing xenophagy by different pathways, namely the inhibition of MTOR or through TRPML1-TFEB activation, on the fate of pathogens that are either susceptible to, evade or require autophagy for intracellular survival. We identified a dose of rapamycin that exclusively induces autophagy through MTOR inhibition and used ML-SA1 to activate the TRPML1-TFEB pathway, which also increases lysosomal biogenesis. We found that ML-SA1 induced greater autophagy flux than rapamycin. By performing in vitro infections with H. pylori, S. Typhimurium, S. flexneri, L. monocytogenes and S. aureus, we established that ML-SA1 had a more potent effect than rapamycin in restricting the growth of pathogens susceptible to xenophagy. In the case of pathogens that produce effectors to block xenophagy, ML-SA1, but not rapamycin, resulted in bacterial killing. During S. aureus infection, which depends on autophagy for intracellular survival, ML-SA1 administration potentiated bacterial growth. We suggest that while targeting the xenophagy pathway holds promise for treatment of intracellular pathogens, a precision approach to select the correct target to induce effective bacterial killing is warranted. Abbreviations: 3-MA: 3-methyladenine, ATG: autophagy-related protein, Baf: bafilomycin A1; Ca2+: calcium, CFU: colony-forming units, DMSO: dimethyl sulfoxide, h: hour, Hp: Helicobacter pylori, hpi: hours post-infection, Lamp1: lysosomal-associated membrane protein 1, LC3: microtubule-associated protein 1A/1B-light chain, Lm: Listeria monocytogenes, LSD: lysosomal storage disorder, min: minutes, mTOR: mechanistic target of rapamycin; mTORC1: mechanistic target of rapamycin complex 1, MEF: mouse embryonic fibroblast, μM: micromolar, moi: multiplicity of infection, nM: nanomolar, OD: optical density, PBS: phosphate buffer saline, Sa: Staphylococcus aureus, SCV: Salmonella containing vacuole, Sifs: Salmonella-induced filaments, Sf: Shigella flexneri, SLAPs: Spacious Listeria containing phagosomes, St: Salmonella Typhimurium TFEB: transcription factor EB, TRPML1: transient receptor potential membrane channel 1, VacA: vacuolating cytotoxin, wt: wild-type.