Rancès Edwige, Voronin Denis, Tran-Van Van, Mavingui Patrick
Université de Lyon, Lyon, France.
J Bacteriol. 2008 Jul;190(14):5020-30. doi: 10.1128/JB.00377-08. Epub 2008 May 23.
A type IV secretion system (T4SS) is used by many symbiotic and pathogenic intracellular bacteria for the successful infection of and survival, proliferation, and persistence within hosts. In this study, the presence and function of the T4SS in Wolbachia strains were investigated by a combination of genetic screening and immunofluorescence microscopy. Two operons of virB-virD4 loci were found in the genome of Wolbachia pipientis strain wAtab3, from the Hymenoptera Asobara tabida, and strain wRi, infecting Drosophila simulans. One operon consisted of five vir genes (virB8, virB9, virB10, virB11, and virD4) and the downstream wspB locus. The other operon was composed of three genes (virB3, virB4, and virB6) and included four additional open reading frames (orf1 to orf4) orientated in the same direction. In cell culture and insect hosts infected with different Wolbachia strains, the bona fide vir genes were polycistronically transcribed, together with the downstream adjacent loci, notably, as virB8 to virD4 and wspB and as virB3, virB4, virB6, and orf1 to orf4. Two peptides encompassing conserved C and N termini of the Wolbachia VirB6 protein were used for the production of polyclonal antibodies. Anti-VirB6 antibodies could detect the corresponding recombinant protein by chemifluorescence on Western blots of total proteins from Escherichia coli transformants and Wolbachia strains cultured in cell lines. Using immunofluorescence microscopy, we further demonstrated that the VirB6 protein was produced by Wolbachia strains in ovaries of insects harboring wAtab3 or wRi and cell lines infected with wAlbB or wMelPop. As VirB6 is known to associate with other VirB proteins to form a membrane-spanning structure, this finding suggests that a T4SS may function in Wolbachia.
许多共生和致病的胞内细菌利用IV型分泌系统（T4SS）成功感染宿主并在宿主体内生存、增殖和持续存在。在本研究中，通过基因筛选和免疫荧光显微镜相结合的方法，研究了沃尔巴克氏体菌株中T4SS的存在和功能。在来自膜翅目昆虫塔比阿索芭拉的沃尔巴克氏体pipientis菌株wAtab3以及感染拟暗果蝇的wRi菌株的基因组中，发现了virB-virD4位点的两个操纵子。一个操纵子由五个vir基因（virB8、virB9、virB10、virB11和virD4）以及下游的wspB位点组成。另一个操纵子由三个基因（virB3、virB4和virB6）组成，并包含四个同向排列的额外开放阅读框（orf1至orf4）。在感染不同沃尔巴克氏体菌株的细胞培养物和昆虫宿主中，真正的vir基因与下游相邻位点一起多顺反子转录，特别是作为virB8至virD4和wspB以及作为virB3、virB4、virB6和orf1至orf4。使用包含沃尔巴克氏体VirB6蛋白保守C端和N端的两种肽来制备多克隆抗体。抗VirB6抗体可以通过化学荧光在来自大肠杆菌转化体和在细胞系中培养的沃尔巴克氏体菌株的总蛋白的蛋白质印迹上检测相应的重组蛋白。使用免疫荧光显微镜，我们进一步证明，VirB6蛋白由携带wAtab3或wRi的昆虫卵巢中的沃尔巴克氏体菌株以及感染wAlbB或wMelPop的细胞系产生。由于已知VirB6与其他VirB蛋白结合形成跨膜结构，这一发现表明T4SS可能在沃尔巴克氏体中发挥作用。
