Study of the function of SPI-22 T6SS from Salmonella bongori in bacterial competition and interactions with phagocytic cells.

Author(s)

Daniel Enrique Sanchez Limache

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2022-03-09

Examining board members

Ethel Bayer Santos; Ana Carolina de Mello Santos Gheller; Marilis do Valle Marques; Cristiano Gallina Moreira

• Advisor: Ethel Bayer Santos

Abstract

Bacteria have evolved antagonistic mechanisms to attack competing species and secure adaptive advantages. The T6SS (type 6 secretion system) is a protein secretion system that functionally resembles a contractile harpoon in which a spear composed of Hcp (hemolysin-coregulated protein), VgrG (valine-glycine repeat protein G) and PAAR (proline-alanine-arginine) proteins is launched toward the target cell to release toxic effectors. In Salmonella spp., the genes responsible for assembling the T6SS are clustered in Salmonella Pathogenicity Islands (SPI). Salmonella bongori carries a SPI-22 that encodes a T6SS unique to this species; however, its function was not yet characterized. The objective of this work was to analyze the function of SPI-22 T6SS from S. bongori regarding its antibacterial and anti-eukaryotic activity. Results demonstrate that SPI-22 T6SS displays antibacterial function. In silico analyses identified several possible effectors secreted by SPI-22 T6SS. Among these effectors, we chose four genes that have VRR-Nuc (virus-type replication-repair nuclease) domain for functional characterization (SBG_1828, SBG_1841, SBG_2718 and SBG_2723). SBG_2723 and SBG_1841, renamed TseV2 and TseV3 (type VI effector with VRR-Nuc), showed toxicity when expressed in Escherichia coli; while SBG_2718 and SBG_1828, renamed TseV1 and TseV4, did not affect the growth of this bacterium. Bacterial competition assays confirmed that TseV2 and TseV3 are secreted via SPI-22 T6SS. Furthermore, bacterial competition assays revealed that VgrG2 (SBG_2715) and VgrG3 (SBG_3770) are important for secretion of TseV2 and TseV3, respectively. To test the anti-eukaryotic activity of SPI-22 T6SS, we performed predation assays using Dictyostelium discoideum to evaluate the ability of this amoeba to form phagocytic plaques once feeding on the wild-type and T6SS mutant strains. Preliminary results suggest that the T6SS mutant may be more susceptible to predation; however, further studies will be needed to clarify this function. In this work we characterized the function of SPI-22 T6SS and identified new effectors secreted by this system, demonstrating the participation of different VgrG proteins in the selection and secretion of these effectors. (AU)

• FAPESP's process: 19/22715-8 - Function of Salmonella bongori SPI-22 T6SS in bacterial competitions and interactions with phagocytic host cells
• Grantee: Daniel Enrique Sánchez Limache
• Support Opportunities: Scholarships in Brazil - Master