Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2
Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2
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DOI：
10.1128/jb.00863-09
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发表时间：
2009-11-15
影响因子：
3.2
通讯作者：
Okada, Nobuhiko
中科院分区：
文献类型：
--
作者：
Miki, Tsuyoshi;Shibagaki, Yoshio;Okada, Nobuhiko
由沙门氏菌致病性岛 2 (SPI-2) 编码的 III 型分泌系统 (T3SS) 参与肠沙门氏菌鼠伤寒血清型的全身感染和细胞内复制。在这项研究中，我们研究了 SsaE 的功能，SsaE 是 SPI-2 基因座内编码的一种小型细胞质蛋白，其结构与 T3SS V 类分子伴侣相似。鼠伤寒沙门氏菌血清型 ssaE 突变体无法分泌 SPI-2 易位蛋白 SseB 和 SPI-2 依赖性效应蛋白 PipB。使用 SsaE-FLAG 融合蛋白进行的免疫共沉淀和质谱分析表明，SsaE 与 SseB 和推定的 T3SS 相关 ATP 酶 SsaN 相互作用。一系列缺失和点突变的 SsaE-FLAG 融合蛋白表明，SsaE 的 C 端卷曲螺旋结构域对于蛋白质-蛋白质相互作用至关重要。尽管据报道 SseA 是 SseB 的伴侣，并且是其在细菌细胞质中的分泌和稳定性所必需的，但当质粒衍生的 SseB 过度表达时，sseA 缺失突变体能够在体外分泌 SseB。相反，ssaE突变株在相同的测定条件下不能将SseB转运至胞外。此外，表达缺乏形成C端卷曲螺旋结构能力的SsaE衍生物的ssaE（I55G）点突变菌株表现出与SPI-2 T3SS无效突变体相当的毒力减弱，表明SsaE的卷曲螺旋相互作用对于功能性SPI-2 T3SS和沙门氏菌毒力来说是绝对必要的。基于这些发现，我们提出 SsaE 识别易位蛋白 SseB 并通过 SPI-2 III 型分泌机制控制其分泌。
The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An S. enterica serovar Typhimurium ssaE mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an sseA deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast, ssaE mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an ssaE(I55G) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for Salmonella virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery.