Label-free quantitative proteomics reveals a role for the Mycobacterium tuberculosis SecA2 pathway in exporting solute binding proteins and Mce transporters to the cell wall

Mycobacterium tuberculosis is an example of a bacterial pathogen with a specialized SecA2-dependent protein export system that contributes to its virulence. Our understanding of the mechanistic basis of SecA2-dependent export and the role(s) of the SecA2 pathway in M. tuberculosis pathogenesis has been hindered by our limited knowledge of the proteins exported by the pathway. Here, we set out to identify M. tuberculosis proteins that use the SecA2 pathway for their export from the bacterial cytoplasm to the cell wall. Using label-free quantitative (LFQ) proteomics involving spectral counting, we compared the cell wall and cytoplasmic proteomes of wild type M. tuberculosis to that of a ΔsecA2 mutant. This work revealed a role for the M. tuberculosis SecA2 pathway in the cell wall localization of solute binding proteins (SBPs) that work with ABC transporters to import solutes. Another discovery was a profound effect of SecA2 on the cell wall localization of the Mce1 and Mce4 lipid transporters, which contribute to M. tuberculosis virulence. In addition to the effects on SBP and Mce transporter export, our LFQ analysis revealed an unexpected relationship between SecA2 and the hypoxia-induced DosR regulon, which is associated with M. tuberculosis latency. Nearly half of the transcriptionally controlled DosR regulon of cytoplasmic proteins were detected at higher levels in the ΔsecA2 mutant versus wild type M. tuberculosis. By increasing the list of M. tuberculosis proteins known to be affected by the SecA2 pathway, this study expands our appreciation of the types of proteins exported by this pathway and guides our understanding of the mechanism of SecA2-dependent protein export in mycobacteria. At the same time, the newly identified SecA2-dependent proteins are helpful for understanding the significance of this pathway to M. tuberculosis virulence and physiology

结核分枝杆菌（Mycobacterium tuberculosis）是一类具备特化SecA2依赖型蛋白质分泌系统（SecA2-dependent protein export system）的细菌病原体，该系统与其毒力密切相关。此前，由于对该通路所转运的蛋白质认知不足，学界对依赖SecA2的蛋白质分泌的机制基础，以及SecA2通路在结核分枝杆菌致病过程中的作用的理解始终受到限制。本研究旨在鉴定结核分枝杆菌中通过SecA2通路从细菌细胞质转运至细胞壁的蛋白质。本研究采用基于光谱计数法的无标记定量（LFQ）蛋白质组学技术，对比了野生型结核分枝杆菌与ΔsecA2突变株的细胞壁蛋白质组与细胞质蛋白质组。本研究揭示，结核分枝杆菌的SecA2通路参与调控溶质结合蛋白（SBPs）的细胞壁定位——这类蛋白质与ABC转运蛋白协同完成溶质的摄取过程。另一项重要发现为，SecA2对Mce1与Mce4脂质转运蛋白的细胞壁定位具有显著调控作用，而这两类转运蛋白可影响结核分枝杆菌的毒力。除上述对溶质结合蛋白与Mce转运蛋白转运的调控作用外，本研究的LFQ蛋白质组学分析还揭示了SecA2与低氧诱导的DosR调节子（DosR regulon）之间的意外关联——该调节子与结核分枝杆菌的潜伏感染密切相关。在经转录调控的DosR调节子所涵盖的细胞质蛋白质中，近半数在ΔsecA2突变株中的表达水平显著高于野生型结核分枝杆菌。本研究新增了一批受SecA2通路调控的结核分枝杆菌蛋白质，拓展了学界对该通路所转运蛋白质种类的认知，同时为理解分枝杆菌属中依赖SecA2的蛋白质分泌机制提供了新的指引。与此同时，本次新鉴定的SecA2依赖型蛋白质，也为阐明该通路在结核分枝杆菌毒力与生理过程中的重要意义提供了关键线索。