Bacteria deliver toxic effectors via type VI secretion systems (T6SSs) to dominate competitors, but the identity and function of many effectors remain unknown. Here we identify a Vibrio antibacterial T6SS effector that contains a previously undescribed, widespread DNase toxin domain that we call PoNe (Polymorphic Nuclease effector). PoNe belongs to a diverse superfamily of PD-(D/E)xK phosphodiesterases, and is associated with several toxin delivery systems including type V, type VI, and type VII. PoNe toxicity is antagonized by cognate immunity proteins (PoNi) containing DUF1911 and DUF1910 domains. In addition to PoNe, the effector contains a domain of unknown function (FIX domain) that is also found N-terminal to known toxin domains and is genetically and functionally linked to T6SS. FIX sequences can be used to identify T6SS effector candidates with potentially novel toxin domains. Our findings underline the modular nature of bacterial effectors harboring delivery or marker domains, specific to a secretion system, fused to interchangeable toxins.

Gram-negative bacteria deliver effectors via the type VI secretion system (T6SS) to outcompete their rivals. Each bacterial strain carries a different arsenal of effectors; the identities of many remain unknown. Here, we present an approach to identify T6SS effectors encoded in bacterial genomes of interest, without prior knowledge of the effectors' domain content or genetic neighborhood. Our pipeline comprises a comparative genomics analysis followed by screening using a surrogate T6SS + strain. Using this approach, we identify an antibacterial effector belonging to the T6SS1 of Vibrio parahaemolyticus, representing a widespread family of T6SS effectors sharing a C-terminal domain that we name Tme (Type VI membranedisrupting effector). Tme effectors function in the periplasm where they intoxicate bacteria by disrupting membrane integrity. We believe our approach can be scaled up to identify additional T6SS effectors in various bacterial genera.

Gram-negative bacteria employ toxin delivery systems to mediate their interactions with neighboring cells. Vibrio parahaemolyticus , an emerging pathogen of humans and marine animals, was shown to deploy antibacterial toxins into competing bacteria via the type VI secretion system (T6SS). Here, we analyzed 1,727 V. parahaemolyticus genomes and revealed the pan-genome T6SS repertoire of this species, including the T6SS gene clusters, horizontally shared auxiliary modules, and toxins.

The type VI secretion system (T6SS) is used by bacteria to deliver toxic effectors directly into target cells. Most T6SSs mediate antibacterial activities, whereas the potential anti-eukaryotic role of T6SS remains understudied. Here, we found a Vibrio T6SS that delivers two novel effectors into mammalian host immune cells. We showed that these effectors induce a pyroptotic cell death in a phagocytosis-dependent manner; we identified the NLRP3 inflammasome as being the underlying mechanism leading to the T6SS-induced pyroptosis. Moreover, we identified a compensatory T6SS-induced pathway that is activated upon inhibition of the canonical pyroptosis pathway. Genetic analyses revealed possible horizontal spread of this T6SS and its anti-eukaryotic effectors into emerging pathogens in the marine environment. Our findings reveal novel T6SS effectors that activate the host inflammasome and possibly contribute to virulence and to the emergence of bacterial pathogens.

The type VI secretion system (T6SS) is deployed by numerous Gramnegative bacteria to deliver toxic effectors into neighbouring cells. The genome of Pantoea agglomerans pv. betae (Pab) phytopathogenic bacteria contains a gene cluster (T6SS1) predicted to encode a complete T6SS. Using secretion and competition assays, we found that T6SS1 in Pab is a functional antibacterial system that allows this pathogen to outcompete rival plant-associated bacteria found in its natural environment. Computational analysis of the T6SS1 gene cluster revealed that antibacterial effector and immunity proteins are encoded within three genomic islands that also harbour arrays of orphan immunity genes or toxin and immunity cassettes. Functional analyses indicated that VgrG, a specialized antibacterial effector, contains a C-terminal catalytically active glucosaminidase domain that is used to degrade prey peptidoglycan. Moreover, we confirmed that a bicistronic unit at the end of the T6SS1 cluster encodes a novel antibacterial T6SS effector and immunity pair. Together, these results demonstrate that Pab T6SS1 is an antibacterial system delivering a lysozyme-like effector to eliminate competitors, and indicate that this bacterium contains additional novel T6SS effectors.Andrea Carobbi and Simone Di Nepi contributed equally to this work.