The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane depolarizing activity is alleviated by co-production of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologues at the tspA locus, suggestive of additional roles in intra-species competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologues.

中文翻译：

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金黄色葡萄球菌VII型分泌系统的膜去极化毒素底物介导种内竞争

VII型蛋白分泌系统（T7SS）在金黄色葡萄球菌菌株中是保守的，并且在毒力和细菌间竞争中起重要作用。迄今为止，只有一种在金黄色葡萄球菌基因组子集中编码的T7SS底物蛋白在功能上得到了表征。在这里，使用无偏蛋白质组学方法，我们将TspA鉴定为另一个T7SS底物。TspA编码远离T7SS基因簇，并在所有金黄色葡萄球菌菌株以及李斯特菌和肠球菌中均发现。来自金黄色葡萄球菌菌株RN6390的TspA的异源表达表明，当其靶向大肠杆菌大肠质时，其C-末端结构域是有毒的，并且使细胞质膜去极化。膜结合的TsaI免疫蛋白的共同产生可减轻膜的去极化活性，在金黄色葡萄球菌染色体上与tspA相邻编码。使用斑马鱼后脑室感染模型，我们证明菌株RN6390的T7SS促进体内细菌复制，而tspA的缺失导致细菌清除率增加。TspA的毒素域是高度多态性的，金黄色葡萄球菌菌株在tspA基因座编码多个tsaI同源物，提示在种内竞争中还有其他作用。一致地，我们证明了在斑马鱼感染模型中由菌株COL对RN6390的TspA依赖性生长抑制，这被TsaI同源物的存在减轻了。TspA的毒素域是高度多态性的，金黄色葡萄球菌菌株在tspA基因座编码多个tsaI同源物，提示在种内竞争中还有其他作用。一致地，我们证明了在斑马鱼感染模型中由菌株COL对RN6390的TspA依赖性生长抑制，这被TsaI同源物的存在减轻了。TspA的毒素域是高度多态性的，金黄色葡萄球菌菌株在tspA基因座编码多个tsaI同源物，提示在种内竞争中还有其他作用。在一致的情况下，我们证明了斑马鱼感染模型中由菌株COL对RN6390的TspA依赖性生长抑制，这可通过TsaI同源物的存在减轻。