| Congresso Brasileiro de Microbiologia 2023 | Resumo: 106-1 | ||||
Resumo:Bacteria evolved several strategies to eliminate competitors from the surrounding environment. The type VI secretion system (T6SS) is a nanomachine present in Gram-negatives that secretes toxic effectors into target competitors. These effectors present an array of biochemical activities to damage membranes, peptidoglycan, and nucleic acids. Previous work described the importance of Salmonella T6SSs in host gut colonization. The T6SS encoded within the SPI-6 (Salmonella Pathogenicity Island 6) is involved in bacterial competition and has only three effectors described to date. The goal of this work was to identify new T6SS effectors in S. enterica. We performed bioinformatic analysis in 10k genomes and identified putative candidates, which were cloned and expressed in Escherichia coli to analyze toxicity. The analysis revealed Tox26 as an evolved effector containing an N-terminal trafficking domain (PAAR) and a C-terminal region with no detectable Pfam domain. Expression of Tox26CT in E. coli showed toxicity in the cytoplasm and a minor effect in the periplasm, which could be neutralized by an immunity gene encoded downstream (Imm26). Bacterial competitions between Salmonella WT or Δt6ss against a double mutant Δtox26/imm26 indicate that Tox26 is secreted via the T6SS. Sequence homology searches revealed that Tox26CT has distant similarity to a predicted protein from the HNH superfamily. Point mutation in conserved His residue (H1544A) reduced toxicity. Colony forming units (CFU) counts before and after Tox26CT expression revealed its bactericidal effect. Time-lapse microscopy of intoxicated E. coli showed that the effector prevents cell division and induces slight cell elongation. E. coli carrying a reporter plasmid with GFP under the control of recA promoter showed that Tox26CT does not activate the SOS response. Plasmids extracted from Tox26CT-expressing bacteria do not show DNA degradation patterns; however, total RNA extraction revealed a band pattern suggesting cleavage or modification of rRNAs. Collectively, these results indicate that Tox26 is an antibacterial T6SS effector that targets rRNAs. Previously described HNH superfamily members display DNase and/or RNase activity. This work offers preliminary data suggesting that Tox26 is the founding member of a novel group of toxins that exclusively targets ribosomal RNA. The characterization of novel protein domains from T6SS effectors hold great potential for the development of biotechnological applications and help understand how bacteria naturally eliminate competitors, possibly revealing new targets to fight antimicrobial resistance. Palavras-chave: Biological conflicts, Nuclease, Salmonella, T6SS Agência de fomento:CAPES and FAPESP | |||||