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 Salmonella typhimurium 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 Salmonella-containing vacuoles (SCVs). The enhanced proteolytic activity due to deployment of lysosomes results in clamping down Salmonella replication in SCVs. Acacetin is effective as a xenophagy compound in an in vivo mouse model of infection and reduces intracellular Salmonella burden.

Abbreviations

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: Salmonella-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.

中文翻译：

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通过恢复 TFEB 介导的异体吞噬来限制细胞内沙门氏菌的复制。

巨自噬/自噬作为先天免疫系统的一部分在清除细胞内病原体方面发挥作用。虽然这个过程是众所周知的，但控制抗菌自噬的机制尚不清楚。在这项研究中，我们表明在细胞内鼠伤寒沙门氏菌在感染后，TFEB（转录因子 EB）（自噬和溶酶体生物发生的主要调节因子）的活性通过在溶酶体上保持磷酸化状态而受到抑制。此外，我们还鉴定了一种新型抗菌小分子自噬（异体自噬）调节剂金合欢素。金合欢素发挥的异体吞噬作用以 MTOR（雷帕霉素激酶的机制靶标）非依赖性、TFEB 依赖性方式发生。金合欢素处理导致细胞核中持续保持活跃的 TFEB，并且还导致 TFEB 介导的功能性溶酶体的诱导，这些溶酶体靶向含沙门氏菌的液泡 (SCV)。由于溶酶体的部署而增强的蛋白水解活性导致抑制沙门氏菌SCV 中的复制。Acacetin在体内感染小鼠模型中作为异种化合物是有效的，并减少细胞内沙门氏菌的负担。

缩写

3-MA：3-甲基腺嘌呤；BafA1：巴弗洛霉素A ₁；CFU：菌落形成单位；DQ-BSA：染料淬灭的牛血清白蛋白；EEA1：早期内体抗原1；FITC：异硫氰酸荧光素；FM 4-64：吡啶鎓，4-(6-[4-{二乙基氨基}苯基]-1,3,5-己三烯基)-1-(3[三乙基氨]丙基)-二溴化物；GFP：绿色荧光蛋白；LAMP1：溶酶体相关膜蛋白 1；MAPILC3/LC3：微管相关蛋白1轻链3；MOI：感染复数；MTOR：雷帕霉素激酶的机制靶点；RFP：红色荧光蛋白；SCV：含沙门氏菌的液泡；SD：标准差；SDS：十二烷基硫酸钠；SEM：标准平均误差；SQSTM1：螯合体 1；TBK1：TANK 结合激酶 1；TFEB：转录因子EB。