Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
J Biol Chem. 2023 Apr;299(4):104589. doi: 10.1016/j.jbc.2023.104589. Epub 2023 Mar 6.
Mycobacterium tuberculosis (Mtb) utilizes sophisticated machinery called the type VII secretion system to translocate virulence factors across its complex lipid membrane. EspB, a ∼36 kDa secreted substrate of the ESX-1 apparatus, was shown to cause ESAT-6-independent host cell death. Despite the current wealth of high-resolution structural information of the ordered N-terminal domain, the mechanism of EspB-mediated virulence remains poorly characterized. Here, we document EspB interaction with phosphatidic acid (PA) and phosphatidylserine (PS) in the context of membranes, through a biophysical approach including transmission electron microscopy and cryo-EM. We were also able to show PA, PS-dependent conversion of monomers to oligomers at physiological pH. Our data suggest that EspB adheres to biological membranes with limited PA and PS. EM of yeast mitochondria with EspB indicates a mitochondrial membrane-binding property of this ESX-1 substrate. Further, we determined the 3D structures of EspB with and without PA and observed plausible stabilization of the low complexity C-terminal domain in the presence of PA. Collectively, our cryo-EM-based structural and functional studies of EspB provide further insight into the host-Mtb interaction.
结核分枝杆菌（Mtb）利用一种称为 VII 型分泌系统的复杂机制，将毒力因子穿过其复杂的脂质膜转运。EspB 是 ESX-1 装置的一种约 36 kDa 的分泌底物，被证明会导致 ESAT-6 非依赖性宿主细胞死亡。尽管目前有大量关于有序 N 端结构域的高分辨率结构信息，但 EspB 介导的毒力机制仍知之甚少。在这里，我们通过包括透射电子显微镜和 cryo-EM 在内的生物物理方法，记录了 EspB 在膜环境中与磷脂酸（PA）和磷脂酰丝氨酸（PS）的相互作用。我们还能够证明在生理 pH 下，PA、PS 依赖性单体向寡聚物的转化。我们的数据表明，EspB 与具有有限 PA 和 PS 的生物膜结合。用 EspB 对酵母线粒体进行的 EM 表明，这种 ESX-1 底物具有线粒体膜结合特性。此外，我们测定了有和没有 PA 的 EspB 的 3D 结构，并观察到在 PA 存在下，低复杂度 C 端结构域的合理稳定化。总之，我们基于 cryo-EM 的 EspB 结构和功能研究为宿主-Mtb 相互作用提供了进一步的见解。
