Michova Hana, Pliva Jan, Jirsova Anezka, Jurnecka David, Kamanova Jana
Laboratory of Infection Biology, Institute of Microbiology, Czech Academy of Sciences, Videnska 1083, Prague, 142 00, Czech Republic.
Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology, Czech Academy of Sciences, Videnska 1083, Prague, 142 00, Czech Republic.
Vet Res. 2025 Jun 8;56(1):113. doi: 10.1186/s13567-025-01548-2.
The type III secretion system (T3SS) is an important virulence factor of Gram-negative bacteria, including the genus Aeromonas, which represents a diverse group of aquatic bacteria. One member of the genus, Aeromonas schubertii, is an emerging pathogen in aquaculture, causing high mortality in snakehead fish. Infections are associated with the formation of white nodules in the internal organs, likely resulting from A. schubertii-induced apoptosis and/or necrosis. The present study investigates the type strain A. schubertii ATCC 43700, which encodes two distinct T3SSs located within Aeromonas pathogenicity islands 1 and 2, referred here to as API1 and API2. We analyzed their role in A. schubertii-induced cytotoxicity and identified novel T3SS effector proteins. Infections of HeLa cells revealed that API1, but not API2, mediates cytotoxicity and induces both apoptotic and necrotic cell death. Moreover, proteomic analysis identified seven candidate effectors secreted by the API1 injectisome. These included two previously described effectors, AopH and AopO from A. salmonicida, as well as five novel effectors named AopI, AopJ, AopL, AopT, and AopU, whose injection into host cells was validated using a split luciferase reporter system. Functional characterization showed that AopL, a homolog of Vibrio parahaemolyticus VopQ, induces caspase-3/-7-independent necrosis, while AopI, a homolog of ExoY from Pseudomonas aeruginosa, suppresses caspase-3/-7 activation and necrosis, revealing a pro-survival function. These results demonstrate the critical role of the API1 injectisome in A. schubertii-induced cytotoxicity and provide experimental identification of novel Aeromonas effectors that cooperate to fine-tune host cell cytotoxicity.
III型分泌系统（T3SS）是革兰氏阴性菌的一种重要毒力因子，包括气单胞菌属，该属代表了一组多样的水生细菌。气单胞菌属的一个成员舒伯特气单胞菌是水产养殖中一种新出现的病原体，可导致乌鳢鱼的高死亡率。感染与内脏中白色结节的形成有关，这可能是由舒伯特气单胞菌诱导的细胞凋亡和/或坏死所致。本研究调查了模式菌株舒伯特气单胞菌ATCC 43700，它编码位于气单胞菌致病岛1和2内的两种不同的T3SS，在此称为API1和API2。我们分析了它们在舒伯特气单胞菌诱导的细胞毒性中的作用，并鉴定了新的T3SS效应蛋白。对HeLa细胞的感染表明，API1而非API2介导细胞毒性并诱导凋亡和坏死性细胞死亡。此外，蛋白质组学分析鉴定了由API1注射体分泌的七种候选效应蛋白。其中包括两种先前描述的效应蛋白，即来自杀鲑气单胞菌的AopH和AopO，以及五种新的效应蛋白，命名为AopI、AopJ、AopL、AopT和AopU，使用分裂荧光素酶报告系统验证了它们注入宿主细胞的情况。功能表征表明，副溶血弧菌VopQ的同源物AopL诱导不依赖于半胱天冬酶-3/-7的坏死，而铜绿假单胞菌ExoY的同源物AopI抑制半胱天冬酶-3/-7的激活和坏死，显示出促生存功能。这些结果证明了API1注射体在舒伯特气单胞菌诱导的细胞毒性中的关键作用，并提供了对协同微调宿主细胞毒性的新型气单胞菌效应蛋白的实验鉴定。
