Cytotoxicity and effector cooperation in the type III secretion system of Aeromonas schubertii

Abstract The type III secretion system (T3SS) is an important virulence factor of Gram-negative bacteria, including the genus Aeromonas , a group of aquatic bacteria capable of both mutualistic and pathogenic interactions. Aeromonas species are increasingly recognized as opportunistic human pathogens. The type strain A. schubertii ATCC 43700 encodes two distinct T3SSs located in the Aeromonas pathogenicity islands 1 and 2, hereby designated as API1 and API2, respectively. Presented work investigates the role of API1 and API2 in A. schubertii -induced cytotoxicity and identifies novel type III secretion effectors. HeLa cell infections showed that API1, but not API2, is essential for cellular cytotoxicity resulting in both apoptotic and necrotic cell death. The ΔAPI1 mutant failed to induce cytotoxicity, whereas the wild-type (WT) and ΔAPI2 strains induced comparable cytotoxic effects. Proteomic analysis identified 7 candidate effectors secreted by the API1 injectisome under low-calcium conditions. These included two previously characterized effectors, AopH and AopO of A. salmonicida , and five novel effectors hereby named AopI, AopJ, AopL, AopT, and AopU, whose injection into host cells via API1 was validated using a split luciferase reporter system. Functional analysis revealed distinct roles for these effectors. AopL, homologous to the VopQ effector of Vibrio parahaemolyticus , accelerated caspase 3-independent necrosis, while AopI, homologous to ExoY of Pseudomonas aeruginosa , suppressed caspase activation and necrosis, indicating a pro-survival function. These results show the role of API1 injectisome in the cytotoxicity of A. schubertii and expand our understanding of T3SS-mediated host- pathogen interactions in Aeromonas species. Importance This work demonstrates that the API1 injectisome is an important cytotoxicity determinant in A. schubertii and identifies novel effectors and their distinct contributions to host cell cytotoxicity, including the pro-survival effect of AopI and the cytotoxic effect of AopL. This interplay highlights a fine-tuned balance between pro-survival and cytotoxic mechanisms which is orchestrated by A. schubertii effectors.