Ma Yue, Keil Verena, Sun Jianjun
From the Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, Texas 79968.
From the Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, Texas 79968
J Biol Chem. 2015 Mar 13;290(11):7314-22. doi: 10.1074/jbc.M114.622076. Epub 2015 Feb 2.
EsxA (ESAT-6), an important virulence factor of Mycobacterium tuberculosis, plays an essential role in phagosome rupture and bacterial cytosolic translocation within host macrophages. Our previous study showed that EsxA exhibits a unique membrane-interacting activity that is not found in its ortholog from nonpathogenic Mycobacterium smegmatis. However, the molecular mechanism of EsxA membrane insertion remains unknown. In this study, we generated truncated EsxA proteins with deletions of the N- and/or C-terminal flexible arm. Using a fluorescence-based liposome leakage assay, we found that both the N- and C-terminal arms were required for membrane disruption. Moreover, we found that, upon acidification, EsxA converted into a more organized structure with increased α-helical content, which was evidenced by CD analysis and intrinsic tryptophan fluorescence. Finally, using an environmentally sensitive fluorescent dye, we obtained direct evidence that the central helix-turn-helix motif of EsxA inserted into the membranes and formed a membrane-spanning pore. A model of EsxA membrane insertion is proposed and discussed.
EsxA（ESAT-6）是结核分枝杆菌的一种重要毒力因子，在宿主巨噬细胞内的吞噬体破裂和细菌胞质转位过程中发挥着关键作用。我们之前的研究表明，EsxA具有独特的膜相互作用活性，这种活性在非致病性耻垢分枝杆菌的同源物中并未发现。然而，EsxA膜插入的分子机制仍然未知。在本研究中，我们生成了缺失N端和/或C端柔性臂的截短型EsxA蛋白。通过基于荧光的脂质体泄漏试验，我们发现N端和C端臂对于膜破坏都是必需的。此外，我们发现，在酸化条件下，EsxA转变为一种α-螺旋含量增加的更有序结构，这通过圆二色性分析和内源色氨酸荧光得以证实。最后，使用一种对环境敏感的荧光染料，我们获得了直接证据，证明EsxA的中央螺旋-转角-螺旋基序插入膜中并形成跨膜孔。我们提出并讨论了EsxA膜插入的模型。
