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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. This 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 cholerae, 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.

INSTRUMENT(S):

ORGANISM(S): Aeromonas Schubertii

SUBMITTER: David Jurnečka

LAB HEAD: Jana Kamanova

PROVIDER: PXD062075 | Pride | 2025-06-10

REPOSITORIES: pride

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			Action	DRS
	Aeromonas_LFQ_Search.xlsx	Xlsx
	Aeromonas_schubertii.fasta	Fasta
	Submission_JK.rar	Other

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Project description:Many Gram-negative bacterial pathogens interact with mammalian cells by using type III secretion systems (T3SS) to inject virulence proteins directly into infected host cells. A subset of these injected protein ‘effectors’ are enzymes that modify the structure and inhibit the function of human proteins by catalyzing the addition of unusual post-translational modifications. T3SS effectors play essential roles in bacterial virulence and their modes of action have provided great insight into the functions and the components of the innate immune system. The E. coli and Citrobacter rodentium NleB effectors, as well as the Salmonella enterica SseK effectors are glycosyltransferases that modify host protein substrates with N-acetyl glucosamine (GlcNAc) on arginine residues. Arginine glycosylation is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles. This post-translational modification disrupts the normal functioning of host immune response proteins. T3SS effectors are chaperoned in the bacterium to keep the effectors partially unfolded and competent for secretion, as well as for targeting the effectors to the T3SS sorting platform. The chaperones are then stripped from their effector substrates at the sorting platform and the effectors are secreted in an unfolded conformation. T3SS effectors are thought to be inactive within the bacterium and fold into their active conformations after they are injected into host cells. While performing mass spectrometry experiments to identify glycosylation substrates of NleB orthologs, we unexpectedly observed that the E. coli glutathione synthetase (GshB) is glycosylated on an arginine residue (R256) by NleB. NleB glycosyltransferase activity is essential to C. rodentium survival in oxidative stress conditions because glycosylation of GshB results in enhanced glutathione production. These data represent, to our knowledge, the first intra-bacterial activity for a T3SS effector and show that effector activities thought to be restricted to host cell compartments additionally play important roles in regulating bacterial physiology.

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

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