| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1078-1 | ||||
Resumo:Gram-negative bacteria have nanomachineries that allow them to change features in their environment. The Type VI secretion system (T6SS) is a dynamic puncturing device that allow the translocation of protein effectors inside prokaryotic or eukaryotic target cells, where they can display toxic effects or counteract the immune system. The T6SS is composed by a sheath-like structure that propels an inner tube of polymerized Hcp and trimers of VgrGs associated to different effectors. We have previously described the role and regulation of the only T6SS of Chromobacterium violaceum, an environmental bacterial pathogen. We found that the primary role of this system is antagonizing other bacteria, favouring the survival of C. violaceum in mixed communities. However, the T6SS antibacterial effector arsenal and its toxic role was still unknown. In this work, we focused our studies in Rearrangement hotspots (Rhs) proteins, polymorphic toxins whose secretion can be T6SS-mediated. In our analysis, we found an Rhs protein (CV_1431) located near VgrG3, outside the main C. violaceum T6SS cluster. Expression of an Rhs C-terminal domain (RhsCT) in Escherichia coli proved to be highly toxic, and this effect was counteracted by expression of its immunity protein, RhsI (CV_1430). Structural prediction of RhsCT by Alpha Fold suggests that it is similar to the catalytic site of ADP-ribosyl-transferases. To test this, we changed an ultra-conserved catalytic glutamic acid to alanine, which decreased RhsCT toxicity. The Rhs secretion dependent on T6SS was established by intraspecies competition assays, in which a strain lacking Rhs and RhsI was sensitive in coculture with parental strain, but not to a strain with inactive T6SS, lacking VgrG3 or Rhs itself. Also, the role of the Rhs toxin as part of the C. violaceum T6SS antibacterial arsenal was evaluated through interspecies competition assays. Deletion of Rhs increased considerably the recovery levels of the target bacteria E. coli and Pseudomonas aeruginosa. Together, these results show that Rhs is an important antibacterial toxin that confers advantage for C. violaceum in bacterial competition. Ongoing studies are focusing on the mode of action and substrate of Rhs. Palavras-chave: Bacterial competition, Polymorphic toxins, Rhs proteins, Type VI secretion system Agência de fomento:FAPESP, FAEPA, CAPES, CNPq | |||||