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Abstract：
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.
摘要：
结核分枝杆菌（Mtb）利用称为VII型分泌系统的复杂机器将毒力因子转移到其复杂的脂质膜上。EspB,ESX-1装置的a~36kDa分泌底物，显示引起ESAT-6非依赖性宿主细胞死亡。尽管当前有序N端域的高分辨率结构信息丰富，EspB介导的毒力机制尚不明确。在这里，我们记录了EspB与磷脂酸（PA）和磷脂酰丝氨酸（PS）在膜环境中的相互作用，通过包括TEM和低温EM在内的生物物理方法。我们还能够展示PA，在生理pH下单体向低聚物的PS依赖性转化。我们的数据表明EspB粘附于具有有限PA和PS的生物膜。具有EspB的酵母线粒体的电子显微镜检查表明该ESX-1底物的线粒体-膜结合特性。Further,我们确定了有和没有PA的EspB的3D结构，并观察到在PA存在下低复杂度C末端结构域的似乎稳定。总的来说，我们对EspB的基于低温EM的结构和功能研究提供了对宿主-Mtb相互作用的进一步了解。
