Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria

Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.

分枝杆菌拥有多种VII型分泌系统（type VII secretion, T7S），可跨越其特殊的细胞包膜分泌蛋白质。其中ESX-5系统仅存在于慢生长分枝杆菌中，负责多种分泌底物的转运。然而，ESX-5底物在细菌生长和/或毒力中的作用目前仍尚不明确。本研究证实，esx-5基因对海分枝杆菌（Mycobacterium marinum）与牛分枝杆菌（Mycobacterium bovis）的生长均为必需。值得注意的是，通过改变外膜的脂质组成，或引入异源孔蛋白MspA（heterologous porin MspA）以提升外膜通透性，可挽救该必需性缺陷。对膜ATP酶EccC5（membrane ATPase EccC5）的首个核苷酸结合结构域进行诱变，可同时阻断ESX-5依赖型分泌与细菌生长，但不影响ESX-5复合物的组装。这提示该挽救效应并非由ESX-5膜复合物形成的孔道所介导，而是源于ESX-5的分泌活性。后续通过蛋白质组学分析鉴定关键ESX-5底物，结果显示细胞表面及细胞包膜组分中所有可检测到的PE和PPE蛋白（PE and PPE proteins）均通过ESX-5途径转运。此外，本研究对野生型海分枝杆菌及表达mspA的菌株开展转座子定向插入位点饱和测序（transposon-directed insertion-site sequencing, TraDIS），以筛选在MspA存在条件下不再必需的基因。该分析进一步验证了esx-5的重要性，但未能鉴定出单一必需的ESX-5底物，表明多种ESX-5底物共同参与介导了该必需功能。最后，对特定碳源培养条件下的表型分析显示，esx-5突变株的疏水性碳源摄取与利用能力显著受损。基于上述实验数据，本研究提出如下模型：ESX-5系统负责转运营养摄取所需的细胞包膜蛋白，这类蛋白可补偿慢生长分枝杆菌中缺乏MspA类孔蛋白的功能缺陷。