Saha Saradindu, Roy Sadhana, Hazra Arnab, Das Debayan, Kumar Vimal, Singh Amit Kumar, Singh Ajay Vir, Mondal Rajesh, Bose Dasgupta Somdeb
Molecular Immunology and Cellular Microbiology Laboratory, Department of Bioscience and Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India.
Laboratory for Animal Experiments, ICMR-National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra 282006, India.
Proc Natl Acad Sci U S A. 2025 Mar 11;122(10):e2404106122. doi: 10.1073/pnas.2404106122. Epub 2025 Mar 4.
The tuberculosis-causing agent (M.tb) establishes its niche inside macrophages by secretion of several virulence factors and engaging many host factors. Mycobacterial infection of macrophages results in a proinflammatory trigger-mediated secretion of TNFα. Protein kinase G (PknG), a Serine/Threonine kinase, is essential for mycobacterial survival within the macrophage. Pathogenic mycobacteria, upon infection, can trigger the secretion of proinflammatory cytokine TNFα, but whether secreted PknG plays any role in TNFα secretion at early stages of infection remains undeciphered. Moreover, at early infection stages, prevention of macrophage apoptosis is vital to successful mycobacterial pathogenesis. Our studies show that mycobacteria-secreted PknG can dampen the expression and concomitant secretion of proinflammatory TNFα. During early infection, M.tb infection-induced generation of reactive nitrogen intermediates (RNI) leads to S-nitrosylation of PknG on Cys109, thereby enabling its secretion into macrophages. Upon M.tb infection, secreted S-nitrosylated PknG phosphorylates macrophage Silencer of Death Domains (SODD) at Thr405, as identified through our phosphoproteomic studies. Thereafter, phosphorylated SODD, through an irreversible binding with the TNFR1 death domain, prevents Caspase8 activation and concomitant extrinsic apoptotic trigger. Moreover, alveolar macrophages from mice infected with PknG-knockout M.tb also exhibited SODD phosphorylation and hindered Caspase8 activation to prevent extrinsic macrophage apoptosis. Therefore, this work exhibits S-nitrosylation-mediated secretion of PknG to induce phosphorylation of macrophage SODD, which, through irreversible interaction with TNFR1, prevented extrinsic macrophage apoptosis at the early stages of infection.
导致结核病的病原体（结核分枝杆菌）通过分泌多种毒力因子并与许多宿主因子相互作用，在巨噬细胞内建立其生存空间。巨噬细胞的分枝杆菌感染会导致促炎触发介导的肿瘤坏死因子α（TNFα）分泌。蛋白激酶G（PknG）是一种丝氨酸/苏氨酸激酶，对分枝杆菌在巨噬细胞内的存活至关重要。致病性分枝杆菌在感染后可触发促炎细胞因子TNFα的分泌，但分泌的PknG在感染早期阶段的TNFα分泌中是否发挥任何作用仍未明确。此外，在感染早期，预防巨噬细胞凋亡对于成功的分枝杆菌发病机制至关重要。我们的研究表明，分枝杆菌分泌的PknG可抑制促炎TNFα的表达和伴随分泌。在感染早期，结核分枝杆菌感染诱导的活性氮中间体（RNI）生成导致PknG的半胱氨酸109发生S-亚硝基化，从而使其能够分泌到巨噬细胞中。通过我们的磷酸蛋白质组学研究确定，在结核分枝杆菌感染后，分泌的S-亚硝基化PknG在苏氨酸405处使巨噬细胞死亡结构域沉默蛋白（SODD）磷酸化。此后，磷酸化SODD通过与肿瘤坏死因子受体1（TNFR1）死亡结构域的不可逆结合，阻止半胱天冬酶8（Caspase8）激活和伴随的外源性凋亡触发。此外，感染PknG基因敲除结核分枝杆菌的小鼠的肺泡巨噬细胞也表现出SODD磷酸化，并阻碍Caspase8激活以防止外源性巨噬细胞凋亡。因此，这项工作展示了S-亚硝基化介导的PknG分泌，以诱导巨噬细胞SODD磷酸化，后者通过与TNFR1的不可逆相互作用，在感染早期阶段防止外源性巨噬细胞凋亡。
