Secretome Analysis Uncovers an Hcp-Family Protein Secreted via a Type VI Secretion System in Agrobacterium tumefaciens
Secretome Analysis Uncovers an Hcp-Family Protein Secreted via a Type VI Secretion System in Agrobacterium tumefaciens
复制标题
DOI：
10.1128/jb.01775-07
复制
发表时间：
2008-02
影响因子：
3.2
通讯作者：
Hung-Yi Wu;P. Chung;Hsiao-Wei Shih;Sy-Ray Wen;E. Lai
中科院分区：
文献类型：
--
作者：
Hung-Yi Wu;P. Chung;Hsiao-Wei Shih;Sy-Ray Wen;E. Lai
农杆菌是一种植物致病菌，能够分泌多种毒力因子进入细胞外空间或宿主细胞。本研究采用散弹枪蛋白组学方法对瘤胃芽孢杆菌的分泌组进行分析，鉴定出12个蛋白，其中包括1个已知的分泌蛋白（VirB1*）和11个潜在的分泌蛋白。有趣的是，一种未知的蛋白，我们命名为溶血素协同调节蛋白（Hcp），是一种预测的可溶性蛋白，没有可识别的n端信号肽。Western blot分析显示，无论在少量培养基还是在丰富培养基中，细胞都能表达和分泌瘤化芽孢杆菌Hcp。进一步的生化和免疫电镜分析表明，细胞内Hcp主要定位于细胞质，小部分定位于膜系统。为了研究农杆菌分泌Hcp的机制，我们产生了一个保守基因icmF缺失的突变体，或者编码最近发现的VI型分泌系统（T6SS）的整个假定操纵子缺失的突变体。Western blot分析表明，在icmF或t6ss操纵子缺失的突变体中，Hcp表达而不分泌到培养基中。在icmF突变体中，Hcp分泌不足被icmF的异源反式表达所补充，这表明Hcp分泌需要icmF。在马铃薯块茎片的肿瘤检测中，hcp的缺失导致肿瘤发生效率降低了约20%至30%，而icmF或t6ss操纵子的缺失则没有观察到一致的差异。这些结果增加了我们对植物和动物致病菌使用的保守T6SS的理解。
ABSTRACT Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant- and animal-pathogenic bacteria.