Global identification of Chromobacterium violaceum T6SS effectors reveals an Rhs antibacterial toxin featuring ADP-ribosyltransferases

ABSTRACT: Bacteria coexist in polymicrobial communities where they engage in complex interactions, including interbacterial antagonism. We recently described that the environmental bacterial pathogen Chromobacterium violaceum has an active type VI secretion system (T6SS), which plays a crucial role in interbacterial competition. However, the arsenal of effectors delivered by this T6SS remains unknown. Here, we identified globally the repertoire of C. violaceum T6SS effectors and characterized a novel effector of Rhs family, which we name RhsF (Rhs with a FIX domain) and its immunity protein, RhsFi. Using mass spectrometry analyses of proteins from culture supernatants and those co-immunoprecipitated with VgrG3, we identified six novel toxin candidates, namely four phospholipases (CV_0012, CV_1234, CV_3971, and CV_3990), a hypothetical protein (CV_2125), and an uncharacterized Rhs protein (CV_1431). Among these T6SS-delivered effectors, we demonstrate that RhsF (CV_1431) was toxic to bacteria in a T6SS dependent manner. The toxicity of the C-terminal domain (RhsF-CT) of RhsF was neutralized upon co-expression with RhsFi (CV_1430), indicating that RhsF-RhsFi composes a toxin-immunity pair. Structure of the RhsF-CT/RhsFi complex solved by X-ray crystallography (1.85 Å resolution) revealed that RhsF exhibits structural features of ADP-ribosyltransferases and provided insights of toxin inhibition via direct occlusion of its catalytic site by RhsFi. Indeed, functional assays confirmed that RhsF toxicity requires a catalytic triad composed by R1403, Y1456, and E1497 residues. Together, our findings uncover the profile of effectors secreted by the T6SS of C. violaceum and establish RhsF as a potent antibacterial toxin, expanding the known repertoire of bacterial arms involved in microbial competition.

INSTRUMENT(S):

ORGANISM(S): Chromobacterium Violaceum Bacteria

SUBMITTER: Matthias Trost  

LAB HEAD: Matthias Trost

PROVIDER: PXD071363 | Pride | 2026-06-01

REPOSITORIES: Pride