Antibacterial type VI secretion system toxins of Salmonella that target nucleic acids

Author(s)

Julia Takuno Hespanhol

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2023-11-28

Examining board members

Ethel Bayer Santos; Waldir Pereira Elias Junior; Frederico José Gueiros Filho; Rodrigo Tavanelli Hernandes

• Advisor: Ethel Bayer Santos

Abstract

Bacteria establish antagonistic relationships to compete for resources in the environment. One of these mechanisms is mediated by the type VI secretion system (T6SS), a contractile apparatus that secretes toxins into target cells after contact between cells. Bacteria that produce toxic effectors also encode cognate immunity proteins to prevent autointoxication and intoxication of sister cells. Salmonella spp. are associated with intestinal infections and encode different T6SSs in distinct pathogenicity islands (SPI-6, SPI-19, SPI-20, SPI-21, SPI-22). These T6SSs are used to compete with the members of the microbiota and establish an infection in the host. The objective of this project was to characterize two new families of antibacterial effectors that target nucleic acids and are secreted by the T6SSs of Salmonella spp. The characterization of the first group of effectors began with the analysis of the Salmonella bongori genome. Using bioinformatics, it was identified a group of effectors secreted by the SPI-22 T6SS called TseVs (type VI secreted effector VRR-Nuc), together with their cognate immunity proteins (TsiVs). Toxicity assays in Escherichia coli revealed that TseV2/TsiV2.1 and TseV3/TsiV3 are effector and immunity protein pairs. Expression of TseV2 and TseV3 in E. coli activated the SOS response and induced the formation of DNA double-strand breaks. Assays with purified recombinant protein revealed that TseV3 is a structure-specific DNase, cleaving substrates that resemble replication fork intermediates or the transcription bubble. The characterization of the second family of effectors started with the analysis of thousands of Salmonella genomes. In Salmonella enterica serotype Oslo, a toxin secreted by the SPI-6 T6SS named STox4 was identified. This protein does not show similarity to domains described in Pfam. Bioinformatics analyzes revealed that STox4 has low similarity with the nuclease superfamily HNH (His-Metal finger). However, no activation of the SOS response in E. coli cells expressing STox4 was observed. Interestingly, changes in the pattern of RNA bands were observed, including the appearance of a smaller band, which suggests that this toxin has RNase activity or is an RNA-modifying protein. STox4 appears to constitute the first member of a new, uncharacterized family of proteins that acts on RNAs. The characterization of effectors secreted by the T6SS expands the repertoire of biological weapons used by pathogens during infections, and reveals new protein domains with undescribed functions. Future studies will be able to identify the biochemical activity of STox4, in addition to identifying its cellular target to determine the mechanism of intoxication and possibly reveal new targets to the development of antimicrobials. (AU)

• FAPESP's process: 22/01364-5 - Functional characterization of a new T6SS antibacterial effector of Salmonella enterica
• Grantee: Julia Takuno Hespanhol
• Support Opportunities: Scholarships in Brazil - Master