Biology subjects

Coulthurst, S.

Publications and source records attributed to Coulthurst, S..

4 recordsLinked to original sources

bioRxiv2026

Tvp complexes support formation of the VgrG-PAAR spike during Type VI secretion system assembly

Type VI secretion systems (T6SSs) are nanomachineries used by bacteria to inject toxic effectors into neighbouring cells during interbacterial competition or infection. The existence of multiple paralogues of the structural proteins VgrG and PAAR and of specific accessory proteins allows the same T6SS to deliver a wide range of effectors. We describe a new set of accessory proteins required for assembly of T6SSs containing the VgrG1-Paar1 spike and delivery of a novel VgrG1-associated membrane-targeting effector in Serratia marcescens. TvpAB, TvpB, TvpC and Paar1 form a pre-complex essential for T6SS assembly, with VgrG1 subsequently replacing TvpC. Phylogenetic analysis and structural modelling reveal that Tvp proteins are widely conserved but Tvp-containing complexes vary in organisation and complexity. We define three classes of Tvp complex, all sharing a DUF2169-containing TvpA protein which stabilises a DUF4150/PAAR-containing protein. Class I, which includes only TvpA, functions without a pre-complex, whereas Classes II and III involve additional Tvp components and two-step assembly. Our findings highlight how modular effector recruitment strategies underlie the versatility of the T6SS and suggest how alternative Tvp systems are tailored to promote assembly of secretion-competent, effector-loaded VgrG-PAAR spikes.

microbiology↗

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

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↗

bioRxiv2024

A widely-occurring family of pore-forming effectors broadens the impact of the Serratia Type VI secretion system

The ability to compete with diverse competitors is essential for bacteria to succeed in microbial communities. A widespread strategy for inter-bacterial competition is the delivery of antibacterial toxins, or effector proteins, directly into rival cells using the Type VI secretion system (T6SS). Whilst a large number of broad-spectrum enzymatic T6SS effectors have been described, relatively few which form pores in target cell membranes have been reported. Here, we describe a widely-occurring new family of T6SS-dependent pore-forming effectors, exemplified by Ssp4 of Serratia marcescens Db10. We show in vitro that Ssp4 forms regulated pores that have higher selectivity for cations and use molecular dynamics simulations to support a high resolution structural model of a tetrameric membrane pore formed by Ssp4. Notably, Ssp4 displays a distinct ion selectivity, phylogenetic distribution and impact on intoxicated cells compared with Ssp6, the other cation-selective pore-forming toxin delivered by the same T6SS. Ssp4 is also active against a wider range of target species than Ssp6, highlighting that T6SS effectors are not always broad-spectrum. Finally, use of Tn-seq to identify Ssp4-resistant mutants reveals that a mucA mutant of Pseudomonas fluorescens, which overproduces extracellular polysaccharide, provides resistance to T6SS attacks. We conclude that possession of two distinct T6SS-dependent pore-forming toxins may be a common strategy to ensure effective de-energisation of closely- and distantly-related competitors.

microbiology↗