<i>Mycobacterium tuberculosis</i> ESX-1-secreted substrate protein EspC promotes mycobacterial survival through endoplasmic reticulum stress-mediated apoptosis

EsxA, secreted by the ESAT-6 secretion system 1 (ESX-1) secretion system, is considered the major Mycobacterium tuberculosis (Mtb) virulence determinant. However, the roles of the individual ESX-1 substrates, such as EspC, remain unclear due to their interdependency for secretion with EsxA. Here, we validated that EspC triggered ER stress-mediated apoptosis in macrophages. The EspC-mediated ER stress was involved in pro-inflammatory cytokines generation, intracellular Ca2+ release, and reactive oxygen species accumulation. Mitochondrial transmembrane potential dissipation and mitochondrial outer membrane permeabilization occurred in EspC-treated macrophages, causing apoptosis. Furthermore, ER stress-mediated apoptosis was effectively induced in EspC-overexpressing Mycobacterium smegmatis-infected macrophages and mice. EspC overexpression caused a significant increase in bacterial survival in the macrophages, spleens, and lungs, and accelerated mouse death was observed. Moreover, the increased viability of bacteria in the macrophages was significantly reduced by pretreatment with the apoptosis inhibitor. Overall, our results revealed that EspC is an essential ESX-1 protein for Mtb–host interactions and EspC-induced ER stress-mediated apoptosis may be employed by Mtb to establish and spread infection. Given the critical roles of the ESX systems in Mtb pathogenesis and immunity, our findings offer new perspectives on the complex host-pathogen interactions and mechanisms underlying ESX-1-mediated pathogenesis.由ESAT-6分泌系统1（ESX-1分泌系统）分泌的EsxA被认为是结核分枝杆菌（Mycobacterium tuberculosis, Mtb）的核心毒力决定因子。然而，由于单个ESX-1底物（如EspC）与EsxA的分泌存在相互依存性，其具体功能仍尚未阐明。本研究证实，EspC可触发巨噬细胞发生内质网应激（endoplasmic reticulum stress, ER stress）介导的细胞凋亡。EspC介导的内质网应激参与了促炎细胞因子的产生、细胞内钙离子释放以及活性氧（reactive oxygen species, ROS）的积累。经EspC处理的巨噬细胞会出现线粒体跨膜电位耗散及线粒体外膜通透性转变，进而引发细胞凋亡。此外，在感染过表达EspC的耻垢分枝杆菌（Mycobacterium smegmatis）的巨噬细胞及小鼠体内，均可有效诱导内质网应激介导的细胞凋亡。EspC过表达可显著提升巨噬细胞、脾脏及肺部的细菌载量，并加速小鼠死亡。进一步研究发现，通过凋亡抑制剂预处理，可显著抑制巨噬细胞内细菌存活率升高的现象。综上，本研究结果揭示EspC是参与结核分枝杆菌与宿主相互作用的关键ESX-1蛋白，且EspC诱导的内质网应激介导的细胞凋亡可能被结核分枝杆菌用于建立并扩散感染。鉴于ESX系统在结核分枝杆菌致病过程与免疫调控中的关键作用，本研究为解析复杂的宿主-病原体相互作用及ESX-1介导的致病机制提供了全新视角。