Browse by author
Lookup NU author(s): Dr Andrew FreyORCiD, Professor Matthias Trost
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© 2026 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license. http://creativecommons.org/licenses/by/4.0/ Bacteria coexist in polymicrobial communities where they engage in complex interactions, including interbacterial antagonism. The environmental bacterial pathogen Chromobacterium violaceum possesses an active type VI secretion system (T6SS), which relies mainly on VgrG3 for its activity and role in interbacterial competition. However, the arsenal of toxic effectors delivered by this T6SS remains unknown. Here, we identify the repertoire of C. violaceum T6SS effectors and characterize a novel antibacterial Rhs-family effector, RhsF (Rhs with a FIX domain), and its cognate immunity protein, RhsFi. Using mass spectrometry analyses of secreted proteins and proteins co-immunoprecipitated with VgrG3, we identified six novel effector candidates, namely four phospholipases, a protein of unknown function, and the previously uncharacterized Rhs protein, RhsF (CV_1431). RhsF contains an N-terminal FIX domain and can intoxicate susceptible bacteria in a T6SS-dependent manner. The action of the C-terminal toxin domain of RhsF (RhsF-CT) is prevented by RhsFi (CV_1430), confirming that RhsF-RhsFi comprises an effector-immunity pair. The structure of the RhsF-CT/RhsFi complex determined by X-ray crystallography (1.85 Å resolution) revealed that RhsF-CT shares structural similarity with ADP-ribosyl transferase toxins and that RhsFi inhibits toxicity via direct occlusion of the RhsF-CT catalytic site. Functional assays indicated that RhsF-CT ADP-ribosylates RNA in vitro and that RhsF toxicity requires a catalytic triad composed of R1403, Y1456, and E1497 residues. Overall, our findings reveal effectors secreted by the T6SS of C. violaceum, establish RhsF as a potent antibacterial toxin, and confirm T6SS-dependent delivery of a FIX-containing Rhs protein, expanding the known repertoire of bacterial arms involved in microbial competition.
Author(s): Alves JA, Pankov G, Frey AM, Trost M, Sgro GG, Coulthurst SJ, da Silva Neto JF
Publication type: Article
Publication status: Published
Journal: Journal of Biological Chemistry
Year: 2026
Volume: 302
Issue: 7
Print publication date: 01/07/2026
Online publication date: 01/06/2026
Acceptance date: 28/04/2026
Date deposited: 15/07/2026
ISSN (print): 0021-9258
ISSN (electronic): 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology Inc.
URL: https://doi.org/10.1016/j.jbc.2026.113216
DOI: 10.1016/j.jbc.2026.113216
Data Access Statement: The crystal structure of the RhsF-CT/RhsFi complex has been deposited into the Protein Data Bank (PDB) under the accession code 9ZDM. The mass spectrometry proteomics data are available in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD071363 for the secretome analysis and PXD071369 for the co-immunoprecipitation of VgrG3. All other data are included within the manuscript or the supporting information.
PubMed id: 42229804
Altmetrics provided by Altmetric
