Restriction of intracellular <i>Salmonella</i> replication by restoring TFEB-mediated xenophagy

Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellular pathogens. Although this process is well known, the mechanisms that control antibacterial autophagy are not clear. In this study we show that during intracellular <i>Salmonella typhimurium</i> infection, the activity of TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, is suppressed by maintaining it in a phosphorylated state on the lysosomes. Furthermore, we have identified a novel, antibacterial small molecule autophagy (xenophagy) modulator, acacetin. The xenophagy effect exerted by acacetin occurs in an MTOR (mechanistic target of rapamycin kinase)-independent, TFEB-dependent manner. Acacetin treatment results in persistently maintaining active TFEB in the nucleus and also in TFEB mediated induction of functional lysosomes that target <i>Salmonella</i>-containing vacuoles (SCVs). The enhanced proteolytic activity due to deployment of lysosomes results in clamping down <i>Salmonella</i> replication in SCVs. Acacetin is effective as a xenophagy compound in an <i>in vivo</i> mouse model of infection and reduces intracellular <i>Salmonella</i> burden. 3-MA: 3-methyladenine; BafA1: bafilomycin A₁; CFU: colony-forming units; DQ-BSA: dye quenched-bovine serum albumin; EEA1: early endosome antigen 1; FITC: fluorescein isothiocyanate; FM 4-64: pyridinium,4-(6-[4-{diethylamino}phenyl]-1,3,5-hexatrienyl)-1-(3[triethylammonio] propyl)-dibromide; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; MAPILC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; RFP: red fluorescent protein; SCVs: <i>Salmonella</i>-containing vacuoles; SD: standard deviation; SDS: sodium dodecyl sulfate; SEM: standard mean error; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TFEB: transcription factor EB.