Section of Molecular Microbiology, Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
Department of Medical Microbiology and Infection Control, Amsterdam UMC, VU Medical Center, Amsterdam, The Netherlands.
mBio. 2022 Dec 20;13(6):e0281922. doi: 10.1128/mbio.02819-22. Epub 2022 Nov 21.
Mycobacteria use specialized type VII secretion systems (T7SSs) to secrete proteins across their diderm cell envelope. One of the T7SS subtypes, named ESX-1, is a major virulence determinant in pathogenic species such as Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. ESX-1 secretes a variety of substrates, called Esx, PE, PPE, and Esp proteins, at least some of which are folded heterodimers. Investigation into the functions of these substrates is problematic, because of the intricate network of codependent secretion between several ESX-1 substrates. Here, we describe the ESX-1 substrate PPE68 as essential for secretion of the highly immunogenic substrates EsxA and EspE via the ESX-1 system in M. marinum. While secreted PPE68 is processed on the cell surface, the majority of cell-associated PPE68 of M. marinum and M. tuberculosis is present in a cytosolic complex with its PE partner and the EspG chaperone. Interfering with the binding of EspG to PPE68 blocked its export and the secretion of EsxA and EspE. In contrast, was not required for the secretion of PPE68, revealing a hierarchy in codependent secretion. Remarkably, the final 10 residues of PPE68, a negatively charged domain, seem essential for EspE secretion, but not for the secretion of EsxA and of PPE68 itself. This indicates that distinctive domains of PPE68 are involved in secretion of the different ESX-1 substrates. Based on these findings, we propose a mechanistic model for the central role of PPE68 in ESX-1-mediated secretion and substrate codependence. Pathogenic mycobacteria, such Mycobacterium tuberculosis and Mycobacterium marinum, use a type VII secretion system (T7SS) subtype, called ESX-1, to mediate intracellular survival via phagosomal rupture and subsequent translocation of the mycobacterium to the host cytosol. Identifying the ESX-1 substrate that is responsible for this process is problematic because of the intricate network of codependent secretion between ESX-1 substrates. Here, we show the central role of the ESX-1 substrate PPE68 for the secretion of ESX-1 substrates in Mycobacterium marinum. Unravelling the mechanism of codependent secretion will aid the functional understanding of T7SSs and will allow the analysis of the individual roles of ESX-1 substrates in the virulence caused by the significant human pathogen Mycobacterium tuberculosis.
分枝杆菌利用专门的 VII 型分泌系统（T7SS）将蛋白穿过双脂细胞膜分泌。T7SS 的一种亚型称为 ESX-1，是结核分枝杆菌和鱼类病原体海分枝杆菌等致病物种的主要毒力决定因子。ESX-1 分泌多种底物，称为 Esx、PE、PPE 和 Esp 蛋白，其中至少一些是折叠的异二聚体。由于 ESX-1 底物之间存在复杂的相互依赖的分泌网络，因此对这些底物的功能进行研究是有问题的。在这里，我们描述了 ESX-1 底物 PPE68 是分枝杆菌 marinum ESX-1 系统中高度免疫原性底物 EsxA 和 EspE 分泌所必需的。虽然分泌的 PPE68 在细胞表面被加工，但分枝杆菌 marinum 和结核分枝杆菌中大多数细胞相关的 PPE68 与它的 PE 伴侣和 EspG 伴侣存在于细胞质复合物中。干扰 EspG 与 PPE68 的结合会阻止其输出和 EsxA 和 EspE 的分泌。相比之下，并不需要 PPE68 的分泌，这揭示了相互依赖的分泌中的层次结构。值得注意的是，PPE68 的最后 10 个残基，一个带负电荷的结构域，对于 EspE 的分泌是必需的，但对于 EsxA 和 PPE68 本身的分泌不是必需的。这表明 PPE68 的不同结构域参与了不同 ESX-1 底物的分泌。基于这些发现，我们提出了一个机制模型，用于解释 PPE68 在 ESX-1 介导的分泌和底物相互依赖中的核心作用。致病性分枝杆菌，如结核分枝杆菌和海分枝杆菌，利用一种称为 ESX-1 的 VII 型分泌系统（T7SS）亚型来通过吞噬体破裂和随后将分枝杆菌转运到宿主细胞质中来介导细胞内生存。由于 ESX-1 底物之间存在复杂的相互依赖的分泌网络，因此确定负责该过程的 ESX-1 底物是有问题的。在这里，我们展示了 ESX-1 底物 PPE68 在分枝杆菌 marinum 中 ESX-1 底物分泌中的核心作用。揭示相互依赖的分泌机制将有助于对 T7SS 的功能理解，并允许分析 ESX-1 底物在重要人类病原体结核分枝杆菌引起的毒力中的个别作用。
