van der Woude Aniek D, Stoop Esther J M, Stiess Michael, Wang Sen, Ummels Roy, van Stempvoort Gunny, Piersma Sander R, Cascioferro Alessandro, Jiménez Connie R, Houben Edith N G, Luirink Joen, Pieters Jean, van der Sar Astrid M, Bitter Wilbert
Department of Medical Microbiology and Infection Control, VU University Medical Center, van der Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands; Department of Molecular Microbiology, Institute of Molecular Cell Biology, VU University, de Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands.
Cell Microbiol. 2014 Feb;16(2):280-95. doi: 10.1111/cmi.12221. Epub 2013 Nov 6.
The pathogenicity of mycobacteria is closely associated with their ability to export virulence factors. For this purpose, mycobacteria possess different protein secretion systems, including the accessory Sec translocation pathway, SecA2. Although this pathway is associated with intracellular survival and virulence, the SecA2-dependent effector proteins remain largely undefined. In this work, we studied a Mycobacterium marinum secA2 mutant with an impaired capacity to initiate granuloma formation in zebrafish embryos. By comparing the proteomic profile of cell envelope fractions from the secA2 mutant with wild type M. marinum, we identified putative SecA2-dependent substrates. Immunoblotting procedures confirmed SecA2-dependent membrane localization for several of these proteins, including the virulence factor protein kinase G (PknG). Interestingly, phenotypical defects of the secA2 mutant are similar to those described for ΔpknG, including phagosomal maturation. Overexpression of PknG in the secA2 mutant restored its localization to the cell envelope. Importantly, PknG-overexpression also partially restored the virulence of the secA2 mutant, as indicated by enhanced infectivity in zebrafish embryos and restored inhibition of phagosomal maturation. These results suggest that SecA2-dependent membrane localization of PknG is an important determinant for M. marinum virulence.
分枝杆菌的致病性与其输出毒力因子的能力密切相关。为此，分枝杆菌拥有不同的蛋白质分泌系统，包括辅助Sec转运途径SecA2。尽管该途径与细胞内存活和毒力相关，但SecA2依赖性效应蛋白在很大程度上仍不明确。在这项研究中，我们研究了一株海分枝杆菌secA2突变体，其在斑马鱼胚胎中引发肉芽肿形成的能力受损。通过比较secA2突变体与野生型海分枝杆菌细胞包膜组分的蛋白质组图谱，我们鉴定出了假定的SecA2依赖性底物。免疫印迹程序证实了其中几种蛋白质（包括毒力因子蛋白激酶G，即PknG）的SecA2依赖性膜定位。有趣的是，secA2突变体的表型缺陷与ΔpknG所描述的相似，包括吞噬体成熟。在secA2突变体中过表达PknG恢复了其在细胞包膜上的定位。重要的是，如斑马鱼胚胎中感染力增强和吞噬体成熟抑制恢复所示，PknG过表达也部分恢复了secA2突变体的毒力。这些结果表明，PknG的SecA2依赖性膜定位是海分枝杆菌毒力的一个重要决定因素。
