Smith Jennifer, Manoranjan Joanna, Pan Miao, Bohsali Amro, Xu Junjie, Liu Jun, McDonald Kent L, Szyk Agnieszka, LaRonde-LeBlanc Nicole, Gao Lian-Yong
Department of Cell Biology and Molecular Genetics and Maryland Pathogens Research Institute, University of Maryland, College Park, Maryland 20742, USA.
Infect Immun. 2008 Dec;76(12):5478-87. doi: 10.1128/IAI.00614-08. Epub 2008 Oct 13.
The ESX-1 secretion system plays a critical role in the virulence of M. tuberculosis and M. marinum, but the precise molecular and cellular mechanisms are not clearly defined. Virulent M. marinum is able to escape from the Mycobacterium-containing vacuole (MCV) into the host cell cytosol, polymerize actin, and spread from cell to cell. In this study, we have examined nine M. marinum ESX-1 mutants and the wild type by using fluorescence and electron microscopy detecting MCV membranes and actin polymerization. We conclude that ESX-1 plays an essential role in M. marinum escape from the MCV. We also show that the ESX-1 mutants acquire the ability to polymerize actin after being artificially delivered into the macrophage cytosol by hypotonic shock treatment, indicating that ESX-1 is not directly involved in initiation of actin polymerization. We provide evidence that M. marinum induces membrane pores approximately 4.5 nm in diameter, and this activity correlates with ESAT-6 secretion. Importantly, purified ESAT-6, but not the other ESX-1-secreted proteins, is able to cause dose-dependent pore formation in host cell membranes. These results suggest that ESAT-6 secreted by M. marinum ESX-1 could play a direct role in producing pores in MCV membranes, facilitating M. marinum escape from the vacuole and cell-to-cell spread. Our study provides new insight into the mechanism by which ESX-1 secretion and ESAT-6 enhance the virulence of mycobacterial infection.
ESX-1分泌系统在结核分枝杆菌和海分枝杆菌的毒力中起着关键作用，但其确切的分子和细胞机制尚不清楚。有毒力的海分枝杆菌能够从含分枝杆菌的液泡（MCV）逃逸到宿主细胞胞质溶胶中，聚合肌动蛋白，并在细胞间传播。在本研究中，我们通过荧光和电子显微镜检测MCV膜和肌动蛋白聚合，对9个海分枝杆菌ESX-1突变体和野生型进行了研究。我们得出结论，ESX-1在海分枝杆菌从MCV逃逸中起重要作用。我们还表明，通过低渗休克处理将ESX-1突变体人工递送至巨噬细胞胞质溶胶后，它们获得了聚合肌动蛋白的能力，这表明ESX-1不直接参与肌动蛋白聚合的起始。我们提供的证据表明，海分枝杆菌诱导直径约4.5nm的膜孔，并且这种活性与ESAT-6分泌相关。重要的是，纯化的ESAT-6，而不是其他ESX-1分泌的蛋白，能够在宿主细胞膜中引起剂量依赖性的孔形成。这些结果表明，海分枝杆菌ESX-1分泌的ESAT-6可能在MCV膜中形成孔、促进海分枝杆菌从液泡逃逸和细胞间传播中起直接作用。我们的研究为ESX-1分泌和ESAT-6增强分枝杆菌感染毒力的机制提供了新的见解。
