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.

• Klíčová slova
• Aeromonas, Aeromonas schubertii, ExoY, VopQ, cytotoxicity, type III secretion system effectors,
• MeSH
• Aeromonas * genetika   patogenita   fyziologie   MeSH
• apoptóza MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• faktory virulence * metabolismus   genetika   MeSH
• gramnegativní bakteriální infekce * mikrobiologie   veterinární   MeSH
• HeLa buňky MeSH
• lidé MeSH
• nemoci ryb * mikrobiologie   MeSH
• sekreční systém typu III * metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• faktory virulence * MeSH
• sekreční systém typu III * MeSH

Bordetella pertussis, the causative agent of whooping cough, is an extracellular, strictly human pathogen. However, it has been shown that B. pertussis cells can escape phagocytic killing and survive in macrophages upon internalization. Our time-resolved RNA-seq data suggest that B. pertussis efficiently adapts to the intramacrophage environment and responds to host bactericidal activities. We show that this adaptive response is multifaceted and, surprisingly, related to the BvgAS two-component system, a master regulator of virulence. Our results show that the expression of this regulatory circuit is downregulated upon internalization. Moreover, we demonstrate that the switch to the avirulent Bvg- phase augments a very complex process based on the adjustment of central and energy metabolism, cell wall reinforcement, maintenance of appropriate redox and metal homeostasis, and repair of damaged macromolecules. Nevertheless, not all observed effects could be simply attributed to the transition to Bvg- phase, suggesting that additional regulators are involved in the adaptation to the intramacrophage environment. Interestingly, a large number of genes required for the metabolism of sulphur were strongly modulated within macrophages. In particular, the mutant lacking two genes encoding cysteine dioxygenases displayed strongly attenuated cytotoxicity toward THP-1 cells. Collectively, our results suggest that intracellular B. pertussis cells have adopted the Bvg- mode to acclimate to the intramacrophage environment and respond to antimicrobial activities elicited by THP-1 cells. Therefore, we hypothesize that the avirulent phase represents an authentic phenotype of internalized B. pertussis cells.

• Klíčová slova
• Bordetella pertussis, BvgAS, adaptation to stress, avirulent phase, cysteine toxicity, intramacrophage environment,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• Bordetella pertussis * metabolismus   MeSH
• fenotyp MeSH
• lidé MeSH
• makrofágy metabolismus   MeSH
• pertuse * MeSH
• regulace genové exprese u bakterií MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH