Functional studies of the Chromobacterium violaceum type VI secretion system

Author(s)

Júlia Aparecida Alves

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2024-05-24

Examining board members

José Freire da Silva Neto; Shaker Chuck Farah; María Eugenia Guazzaroni; Frederico José Gueiros Filho

• Advisor: José Freire da Silva Neto

Abstract

Bacteria have secretion systems for the translocation of proteins, such as toxins and other virulence factors, across the cell membrane to the external environment or into target cells. The type VI secretion system (T6SS) is a contractile machinery of Gram-negative bacteria that fires effector proteins directly into target cells. Secretion occurs by contraction of the VipAB sheath, which leads to the propulsion of the Hcp tube to which VgrG/PAAR proteins and effectors are associated. The T6SS secretes antibacterial toxins during competition between bacteria and effectors that subvert host cell functions. In this work, we characterized the T6SS of Chromobacterium violaceum, a Gram-negative environmental bacterium and opportunistic pathogen in humans. We identified in the C. violaceum genome a main gene cluster and smaller clusters encoding the components of the T6SS. The functionality of the T6SS was evaluated by fluorescence microscopy of sfGFP fused to VipA, by Hcp secretion, and by competition assays against other bacteria, using wild-type C. violaceum and T6SS mutants. The data indicated that the T6SS is active in laboratory culture conditions at low cell density, essential in the competition of C. violaceum against other bacteria, but dispensable for virulence in mice. T6SS regulation was studied by analyzing the activity of promoters of the main cluster. Our data indicated that transcriptional regulation depends on growth phase and CviR, the global regulator of quorum sensing (QS). Consistent with these data, deletion of cviR reduced the production and abolished the secretion of Hcp, in addition to ceasing T6SS firing and strongly reducing the ability of C. violaceum to compete against other bacteria. Mutation of cviI, which encodes the enzyme that produces QS inducers, practically did not affect the T6SS, indicating a non-canonical role of the QS system CviIR in the T6SS. To identify the arsenal of effectors of the C. violaceum T6SS, proteomic analyzes were carried out by mass spectrometry comparing the supernatant of the wild-type strain with the supernatant of a T6SS mutant, in addition to analysis by co-immunoprecipitation of VgrG3. These assays allow to identify as T6SS effectors three phospholipases from the Tle1 family, one from the Tle5 family, a hypothetical protein (CV_2125), and an Rhs protein (CV_1431), named RhsF (Rhs-containing FIX motif). Proteins from the Rhs family are polymorphic toxins. We demonstrated that RhsF is an antibacterial toxin, as controlled expression of its C-terminal domain (RhsF-CT) caused the death of Escherichia coli. Co-expression of RhsF-CT with RhsI, a protein encoded by the gene following rhsF, abolished RhsF toxicity in E. coli, indicating that RhsFI compose a toxin/antitoxin pair. Intraspecies competition assays against strains with rhsFI deletion confirmed that RhsF is secreted by the T6SS, and interspecies competition assays showed that RhsF is indeed an important effector in competition against other bacteria. Analysis of the predicted structure of RhsF and the structure determined by X-ray crystallography of the RhsF-CT/RhsI complex indicates that RhsF is possibly an ADP-ribosyltransferase, whose toxic activity is neutralized by RhsI via obstruction of the catalytic site of the toxin. Assays with biotinylated NAD+ suggest that RhsF ribosylates proteins. Together, the results of this work allowed us to determine the importance of the T6SS in the biology of C. violaceum, identify several T6SS effectors, and characterize a new antibacterial toxin/antitoxin pair from the Rhs family. (AU)

• FAPESP's process: 18/03979-1 - Functional studies of the Chromobacterium violaceum type VI secretion system
• Grantee: Júlia Aparecida Alves
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)