PDF(Pubmed)
Abstract：
Mycobacteria utilize type VII secretion systems (T7SSs) to secrete proteins across their highly hydrophobic and diderm cell envelope. Pathogenic mycobacteria have up to five different T7SSs, called ESX-1 to ESX-5, which are crucial for growth and virulence. Here, we use a functionally reconstituted ESX-5 system in the avirulent species Mycobacterium smegmatis that lacks ESX-5, to define the role of each esx-5 gene in system functionality. By creating an array of gene deletions and assessing protein levels of components and membrane complex assembly, we observed that only the five components of the inner membrane complex are required for its assembly. However, in addition to these five core components, active secretion also depends on both the Esx and PE/PPE substrates. Tagging the PPE substrates followed by subcellular fractionation, surface labeling and membrane extraction showed that these proteins localize to the mycobacterial outer membrane. This indicates that they could play a role in secretion across this enigmatic outer barrier. These results provide the first full overview of the role of each esx-5 gene in T7SS functionality. IMPORTANCE Pathogenic mycobacteria, such as the notorious Mycobacterium tuberculosis, are highly successful as pathogens, in part due to their specific and diderm cell envelope, with a mycolic acid-containing outer membrane. The architecture of this highly impermeable membrane is little understood and the proteins that populate it even less so. To transport proteins across their cell envelope, mycobacteria employ a specialized transport pathway called type VII secretion. While recent studies have elucidated the type VII secretion membrane channel that mediates transport across the inner membrane, the identity of the outer membrane channel remains a black box. Here, we show evidence that specific substrates of the type VII pathway could form these channels. Elucidating the pathway and mechanism of protein secretion through the mycobacterial outer membrane will allow its exploitation for the development of novel mycobacterial therapeutics.
摘要：
分枝杆菌利用VII型分泌系统（T7SS）通过其高度疏水性和表皮细胞包膜分泌蛋白质。致病性分枝杆菌有多达五种不同的T7SS，称为ESX-1至ESX-5，它们对生长和毒力至关重要。这里,我们在缺乏ESX-5的抗毒分枝杆菌中使用功能重建的ESX-5系统，以定义每个esx-5基因在系统功能中的作用。通过创建一系列基因缺失并评估成分和膜复合物组装的蛋白质水平，我们观察到，只有五个组成部分的内膜复合体是必需的组装。然而,除了这五个核心组成部分，活性分泌还取决于Esx和PE/PPE底物。标记PPE底物，然后进行亚细胞分馏，表面标记和膜提取表明，这些蛋白质定位于分枝杆菌外膜。这表明它们可以在这个神秘的外部屏障的分泌中发挥作用。这些结果提供了每个esx-5基因在T7SS功能中的作用的第一个完整概述。重要性致病性分枝杆菌，比如臭名昭著的结核分枝杆菌，作为病原体非常成功，部分原因是它们的特殊和表皮细胞包膜，外膜含霉菌酸.这种高度不可渗透的膜的结构鲜为人知，而填充它的蛋白质则更少。为了运输蛋白质穿过它们的细胞包膜，分枝杆菌采用称为VII型分泌的专门转运途径。虽然最近的研究已经阐明了介导跨内膜运输的VII型分泌膜通道，外膜通道的身份仍然是一个黑盒子。这里,我们证明了VII型途径的特定底物可以形成这些通道。阐明通过分枝杆菌外膜分泌蛋白质的途径和机制将允许其用于开发新的分枝杆菌疗法。
