Biology subjects

Pankov, G.

Publications and source records attributed to Pankov, G..

3 recordsLinked to original sources

bioRxiv2025

Global identification of Chromobacterium violaceum T6SS effectors reveals an Rhs antibacterial toxin featuring FIX and ADP-ribosyltransferase domains

Bacteria coexist in polymicrobial communities where they engage in complex interactions, including interbacterial antagonism. We recently reported 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 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 was shown to intoxicate susceptible bacteria in a T6SS-dependent manner. The action of the C-terminal toxin domain of RhsF (RhsF-CT) is prevented in the presence of 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 [A] resolution) revealed that RhsF-CT shares structural similarity with ADP-ribosyltransferase toxins and that RhsFi inhibits toxicity via direct occlusion of the RhsF-CT catalytic site. Functional assays confirmed that RhsF toxicity requires a catalytic triad composed of R1403, Y1456, and E1497 residues. Overall, our findings reveal the 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.

biochemistry↗

bioRxiv2025

The Type VI Secretion System Antifungal Effector Tfe2 Inhibits Protein Translation and Drives Hyperactivation of TORC1.

Type VI Secretion Systems (T6SS) are utilised by many bacteria to deliver toxic effectors into neighbouring bacterial, fungal or host cells. Whilst many antibacterial effectors are well characterised, much less is known regarding the identity or mode-of-action of antifungal effectors. Here we combine structural modelling with proteomics and in vivo approaches, to show that the Serratia marcescens antifungal effector Tfe2 adopts a novel fold and functions as a potent inhibitor of protein translation leading to hyperactivation of the TORC1 kinase. We show that Tfe2 expression in Saccharomyces cerevisiae, or treatment with the protein translation inhibitor cycloheximide, drive identical increases in free intracellular amino acids and hyperactivation of TORC1. This, in turn, triggers the Tfe2 and cycloheximide-mediated rapid turnover of amino acid transporters through stimulating substrate-independent endocytosis. Polysome profiling, however, revealed differences in Tfe2 and cycloheximide-mediated protein translation inhibition, with Tfe2 inhibiting initiation of translation. Tfe2-mediated hyperactivation of TORC1 may also underpin adaptive responses to this effector which include significant remodelling of the lipidome and notable alterations in organelle and cell wall structures. Collectively this study has provided new insight into the mode-of-action of a structurally novel antifungal effector Tfe2.

microbiology↗

bioRxiv2025

An Rhs effector uses distinct target cell functions to intoxicate bacterial and fungal competitors

Many bacteria use Type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighbouring bacterial or fungal cells as a means of inter-microbial competition. Compared with numerous antibacterial effectors, few antifungal effectors have been described. Furthermore, how T6SS-delivered effectors reach their site of action in different types of target cell remains poorly understood. Here, we combine structural biology with in vivo approaches to show that Rhs2 from Serratia marcescens Db10 is a dual-kingdom T6SS-dependent DNase effector which hijacks distinct, essential target cell functions in order to reach its site of action in bacterial and fungal cells. In bacterial cells, the Rhs2 toxin domain (Rhs2CT) interacts specifically with the elongation factor, EF-Tu, and, in sibling cells, interacts with the cognate immunity protein in an unusual manner. Interaction with EF-Tu is essential for T6SS-mediated intoxication of bacterial cells by Rhs2, but not for DNase activity or intoxication of fungal cells, implying it facilitates entry of Rhs2CT across the inner membrane to the cytoplasm. Alternatively, in fungal cells, Rhs2CT translocates to the nucleus using the nuclear import machinery. Our findings reveal how a single effector domain can act against targets with distinct cellular architectures and suggest that dual-kingdom effectors may occur widely.

microbiology↗