Characterization of Rhs effectors related to the Chromobacterium violaceum type VI secretion system

• Grant number: 22/01586-8
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: October 01, 2022
• End date: September 30, 2023
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: José Freire da Silva Neto
• Grantee: Júlia Aparecida Alves
• Supervisor: Sarah Jane Coulthurst
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: University of Dundee, Scotland
• Associated to the scholarship: 18/03979-1 - Functional studies of the Chromobacterium violaceum type VI secretion system, BP.DD

Abstract

The type VI secretion system (T6SS) is a complex nanomachine used by Gram-negative bacteria to deliver effector proteins into target cells. Although some T6SS effectors have anti-host and anti-fungi activity, the T6SS is mainly involved in interbacterial competition through the secretion of antibacterial effectors. Chromobacterium violaceum is a free-living, saprophytic, Gram-negative ²-proteobacterium that can act as an opportunistic pathogen in mammals. We found that C. violaceum encodes a functional T6SS machinery active under laboratory conditions that has pivotal importance for competition against competing bacteria, such as Pseudomonas aeruginosa, Escherichia coli, and Stenotrophomonas maltophilia. However, it is still unclear which T6SS effectors are responsible for target intoxication, and their toxic activities are unknown. We previously selected through in-silico analysis 12 effector candidates for cloning, expressing, and evaluating their cytotoxicity in E. coli cells. In our analysis, we found four Rhs-repeat-containing proteins, a class of polymorphic toxins commonly associated with the T6SS that share a core section rich in Rhs/YD repetitions and a non-conserved C-terminal with toxic activity. The Rhs proteins selected to be characterized in this project (CV_1429, CV_1431, CV_1238, and CV_1239) are encoded by genes located in orphan vgrG gene clusters, a feature of Rhs secreted by T6SSs. Although these Rhs have canonical core sequences with Rhs repeats, they lack typical N-terminal domains for T6SS secretion. Moreover, these Rhs have potentially novel toxic activities, given that their C-terminal regions lack any known or predicted toxin domain. We propose in this project to study these Rhs proteins using genetic and biochemical approaches to characterize their toxic domains and their impact on interbacterial competition. (AU)