ABSTRACT

The Mycobacterium ulcerans exotoxin, mycolactone, is responsible for the immunosuppression and tissue necrosis that characterizes Buruli ulcer. Mycolactone inhibits SEC61-dependent co-translational translocation of proteins into the endoplasmic reticulum and the resultant cytosolic translation triggers degradation of mislocalized proteins by the ubiquitin-proteasome system. Inhibition of SEC61 by mycolactone also activates multiple EIF2S1/eIF2α kinases in the integrated stress response (ISR). Here we show mycolactone increased canonical markers of selective macroautophagy/autophagy LC3B-II, ubiquitin and SQSTM1/p62 in diverse disease-relevant primary cells and cell lines. Increased formation of puncta positive for the early autophagy markers WIPI2, RB1CC1/FIP200 and ATG16L1 indicates increased initiation of autophagy. The mycolactone response was SEC61A1-dependent and involved a pathway that required RB1CC1 but not ULK. Deletion of Sqstm1 reduced cell survival in the presence of mycolactone, suggesting this response protects against the increased cytosolic protein burden caused by the toxin. However, reconstitution of baseline SQSTM1 expression in cells lacking all autophagy receptor proteins could not rescue viability. Translational regulation by EIF2S1 in the ISR plays a key role in the autophagic response to mycolactone. Mycolactone-dependent induction of SQSTM1 was reduced in eif2ak3^−/-/perk^−/- cells while the p-EIF2S1 antagonist ISRIB reversed the upregulation of SQSTM1 and reduced RB1CC1, WIPI2 and LC3B puncta formation. Increased SQSTM1 staining could be seen in Buruli ulcer patient skin biopsy samples, reinforcing genetic data that suggests autophagy is relevant to disease pathology. Since selective autophagy and the ISR are both implicated in neurodegeneration, cancer and inflammation, the pathway uncovered here may have a broad relevance to human disease.

Abbreviations: ATF4: activating transcription factor 4; ATG: autophagy related; BAF: bafilomycin A₁; ATG16L1: autophagy related 16 like 1; BU: Buruli ulcer; CQ: chloroquine; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; CALCOCO2: calcium binding and coiled-coil domain 2; DMSO: dimethyl sulfoxide; EIF2S1: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; GFP: green fluorescent protein; HDMEC: human dermal microvascular endothelial cells; HFFF: human fetal foreskin fibroblasts; ISR: integrated stress response; ISRIB: integrated stress response inhibitor; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; Myco: mycolactone; NBR1: NBR1 autophagy cargo receptor; NFE2L2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PFA: paraformaldehyde; PtdIns3P: phosphatidylinositol-3-phosphate; RB1CC1: RB1-inducible coiled coil 1; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; ULK: unc-51 like autophagy activating kinase; UPS: ubiquitin-proteasome system; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type.

摘要

溃疡分枝杆菌外毒素分枝杆菌内酯负责免疫抑制和组织坏死，这是布鲁里溃疡的特征。Mycolactone 抑制 SEC61 依赖的蛋白质共翻译易位到内质网中，由此产生的胞质翻译触发了泛素-蛋白酶体系统对错误定位的蛋白质的降解。分枝杆菌内酯对 SEC61 的抑制也会激活综合应激反应 (ISR) 中的多种 EIF2S1/eIF2α 激酶。在这里，我们展示了在多种疾病相关的原代细胞和细胞系中，mycolactone 增加了选择性巨自噬/自噬 LC3B-II、泛素和 SQSTM1/p62 的经典标志物。早期自噬标志物 WIPI2、RB1CC1/FIP200 和 ATG16L1 阳性斑点形成的增加表明自噬的启动增加。霉菌内酯反应是 SEC61A1 依赖性的，并且涉及需要 RB1CC1 但不需要 ULK 的途径。删除Sqstm1在存在霉内酯的情况下降低了细胞存活率，表明这种反应可以防止由毒素引起的细胞溶质蛋白负荷增加。然而，在缺乏所有自噬受体蛋白的细胞中重建基线 SQSTM1 表达并不能挽救活力。ISR 中 EIF2S1 的翻译调节在对霉内酯的自噬反应中起关键作用。SQSTM1 的 Mycolactone 依赖性诱导在 eif2ak3 中降低- ^/- /perk ^-/-而 p-EIF2S1 拮抗剂 ISRIB 逆转了 SQSTM1 的上调并减少了 RB1CC1、WIPI2 和 LC3B 斑点的形成。在布鲁里溃疡患者皮肤活检样本中可以看到 SQSTM1 染色增加，这加强了表明自噬与疾病病理学相关的遗传数据。由于选择性自噬和 ISR 都与神经退行性变、癌症和炎症有关，因此这里发现的途径可能与人类疾病有广泛的相关性。

缩写： ATF4：激活转录因子 4；ATG：自噬相关；BAF：巴弗洛霉素A ₁; ATG16L1：自噬相关 16 like 1；BU：布鲁里溃疡；CQ：氯喹；EIF2AK3：真核翻译起始因子 2 α 激酶 3；CALCOCO2：钙结合和卷曲螺旋结构域 2；DMSO：二甲亚砜；EIF2S1：真核翻译起始因子 2 亚基 alpha；ER：内质网；GFP：绿色荧光蛋白；HDMEC：人真皮微血管内皮细胞；HFFF：人胎儿包皮成纤维细胞；ISR：综合压力反应；ISRIB：综合应激反应抑制剂；MAP1LC3B/LC3B：微管相关蛋白1轻链3β；MEF：小鼠胚胎成纤维细胞；Myco：霉内酯；NBR1：NBR1 自噬货物受体；NFE2L2：核因子，红系2样2；OPTN：optineurin；PFA：多聚甲醛；PtdIns3P：3-磷酸磷脂酰肌醇；RB1CC1：RB1-感应线圈1；SQSTM1：螯合体 1；TAX1BP1：Tax1 结合蛋白 1；ULK：unc-51 样自噬激活激酶；UPS：泛素-蛋白酶体系统；WIPI：WD重复结构域，磷酸肌醇相互作用；WT：野生型。