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.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health challenge, primarily due to the increasing prevalence of drug resistance. Consequently, the development of drugs with novel modes of action (MOAs) is urgently required. In this study, we discovered and characterized two potent inhibitors, Pi-1-58 and Pi-2-26, targeting the prokaryotic ubiquitin-like protein (Pup) ligase PafA of Mtb. Pi-1-58 was identified through computer-aided drug screening. The binding mode of Pi-1-58 and PafA was investigated through molecular docking, followed by experimental validations. Based on the core structure of Pi-1-58, we developed a more potent inhibitor, Pi-2-26, through structure-guided drug design. Both Pi-1-58 and Pi-2-26 exhibited selective and specific inhibition of PafA according to biochemical and cell-based assays. Importantly, the inhibitors demonstrated significant inhibition on Mtb survival in the presence of nitric oxide, mimicking the in vivo nitrogen limited environment that Mtb encountered in macrophage. Our findings provide a comprehensive understanding of the structural and functional aspects of these PafA inhibitors and establish a solid foundation for the development of novel therapeutics against tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major global health threat. A key virulence mechanism involves the pathogen’s ability to inhibit apoptosis in infected macrophages, thereby facilitating immune evasion and persistent infection. The pro-apoptotic protein Bcl-2-interacting killer (BIK) represents a critical mediator of macrophage apoptosis. In this study, we engineered a recombinant Mycobacterium smegmatis strain (rMs-BIK) designed to secrete BIK via fusion with the Ag85B signal peptide (ASP). Secretion and expression of the BIK protein were confirmed by Western blotting. Quantitative flow cytometric analysis revealed that rMs-BIK induced significant apoptosis in RAW264.7 macrophages, with rates of 14.49%, 14.22%, and 22.24% at 12, 24, and 48 h post-infection, respectively. These values were markedly higher than those observed in cells infected with the wild-type strain (7.68%, 9.24%, and 10.90%) or the empty vector control (8.80%, 11.19%, and 14.43%). Our findings demonstrate the feasibility of functional pro-apoptotic protein delivery using a mycobacterial vector, providing a robust foundation for the future development of recombinant BCG-based vaccine candidates.