Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC.
Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC.
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DOI：
10.1099/mic.0.021865-0
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发表时间：
2009-04
期刊：
影响因子：
1.5
通讯作者：
Bo Wang;Z. Mo;Y. Mao;Y. Zou;P. Xiao;J. Li;J. Yang;X. Ye;K. Leung;Pei-jun Zhang
中科院分区：
文献类型：
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作者：
Bo Wang;Z. Mo;Y. Mao;Y. Zou;P. Xiao;J. Li;J. Yang;X. Ye;K. Leung;Pei-jun Zhang
迟缓爱德华氏菌是一种重要的革兰氏阴性肠道病原体，影响动物和人类。它具有致病所必需的III型分泌系统（T3 SS）。EseB、EseC和EseD已显示在分泌后形成易位子复合物，而EscC用作EseB和EseD的T3 SS伴侣。在本文中，我们确定EscA，所需的蛋白质的积累和适当分泌的另一个易位子组件，EseC。escA基因位于eseC的上游，EscA蛋白具有T3 SS分子伴侣的特征。细胞分级实验表明，EscA位于细胞质和细胞质膜上。突变与框内缺失的escA大大降低EseC的分泌，而escA的互补恢复野生型分泌表型。在EscA不存在的情况下，EseC在细胞质中的稳定和积累也受到影响。escA的突变不影响eseC的转录，但降低了EseC的积累水平，如通过使用EseC-LacZ融合蛋白在Ed. tarda中测量的。共纯化和免疫共沉淀研究表明EscA和EseC之间存在特异性相互作用。进一步的分析表明，EseC的残基31-137是EseC-EscA相互作用所必需的。EseC残基31-137的突变减少了EseC在迟缓爱德华中的分泌和积累。最后，感染实验表明，突变的EscA和残基31-137的EseC增加LD（50）在蓝色gourami鱼约10倍。这些结果表明，EscA作为EseC的特异性伴侣发挥作用，并有助于Ed. tarda的毒力。
Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and co-immunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31-137 of EseC are required for EseC-EscA interaction. Mutation of EseC residues 31-137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31-137 of EseC increased the LD(50) by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.