The type VI secretion system (T6SS) is crucial in interbacterial competition and is a virulence determinant of many Gram-negative bacteria. Several T6SS effectors are covalently fused to secreted T6SS structural components such as the VgrG spike for delivery into target cells. In Pseudomonas aeruginosa, the VgrG2b effector was previously proposed to mediate bacterial internalization into eukaryotic cells. In this work, we find that the VgrG2b C-terminal domain (VgrG2bC-ter) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein. We resolve the structure of VgrG2bC-ter and confirm it is a member of the zinc-metallopeptidase family of enzymes. We show that this effector causes membrane blebbing at midcell, which suggests a distinct type of T6SS-mediated growth inhibition through interference with cell division, mimicking the impact of β-lactam antibiotics. Our study introduces a further effector family to the T6SS arsenal and demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells.

The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many Proteobacteria to target effectors/toxins into both eukaryotic and prokaryotic cells. We report that Agrobacterium tumefaciens, a soil bacterium that triggers tumorigenesis in plants, produces a family of type VI DNase effectors (Tde) that are distinct from previously known polymorphic toxins and nucleases. Tde exhibits an antibacterial DNase activity that relies on a conserved HxxD motif and can be counteracted by a cognate immunity protein, Tdi. In vitro, A. tumefaciens T6SS could kill Escherichia coli but triggered a lethal counterattack by Pseudomonas aeruginosa upon injection of the Tde toxins. However, in an in planta coinfection assay, A. tumefaciens used Tde effectors to attack both siblings cells and P. aeruginosa to ultimately gain a competitive advantage. Such acquired T6SS-dependent fitness in vivo and conservation of Tde-Tdi couples in bacteria highlights a widespread antibacterial weapon beneficial for niche colonization.

Bacterial type VI secretion systems (T6SSs) are contractile nanomachines that deliver proteinic substrates into target prokaryotic or eukaryotic cells and the surrounding milieu. The genus Campylobacter encompasses 39 recognized species and 13 subspecies, with many belonging to a group known as ‘emerging Campylobacter pathogens’. Within Campylobacter , seven species have been identified to harbour a complete T6SS cluster but have yet to be comparatively assessed. In this study, using systematic bioinformatics approaches and the T6SS-positive Campylobacter jejuni 488 strain as a reference, we explored the genus-wide prevalence, similarity and make-up of the T6SS amongst 372 publicly available ‘complete’ Campylobacter genomes. Our analyses predict that approximately one-third of Campylobacter species possess a T6SS. We also putatively report the first identification of a T6SS in four species: Campylobacter cuniculorum, Campylobacter helveticus, Campylobacter armoricus and Campylobacter ornithocola . The Campylobacter T6SSs cluster into three distinct organizations (I–III), of which two break down into further variants. Thirty T6SS-containing genomes were found to harbour more than one vgrG gene, with Campylobacter lari strain NCTC 11845 possessing five. Analysis of the C. jejuni Pathogenicity Island-1 confirmed its conservation amongst T6SS-positive C. jejuni strains, as well as highlighting its diverse genetic composition, including additional putative effector–immunity pairs (e.g. PoNe and DUF1911 domains). Effector–immunity pairs were also observed neighbouring vgrGs in several other Campylobacter species, in addition to putative genes encoding nucleases, lysozymes, ATPases and a ferric ATP-binding cassette uptake system. These observations highlight the diverse genetic make-up of the T6SS within Campylobacter and provide further evidence of its role in pathogenesis.

The role of the Type VI secretion system (T6SS) in Campylobacter jejuni is poorly understood despite an increasing prevalence of the T6SS in recent C. jejuni isolates in humans and chickens. The T6SS is a contractile secretion machinery capable of delivering effectors that can play a role in host colonization and niche establishment. During host colonization, C. jejuni is exposed to oxidative stress in the host gastrointestinal tract, and in other bacteria the T6SS has been linked with the oxidative stress response. In this study, comparisons of whole genome sequences of a novel human isolate 488 with previously sequenced strains revealed a single highly conserved T6SS cluster shared between strains isolated from humans and chickens. The presence of a functional T6SS in the 488 wild-type strain is indicated by expression of T6SS genes and secretion of the effector TssD. Increased expression of oxidative stress response genes katA, sodB, and ahpC, and increased oxidative stress resistance in 488 wild-type strain suggest T6SS is associated with oxidative stress response. The role of the T6SS in interactions with host cells is explored using in vitro and in vivo models, and the presence of the T6SS is shown to increase C. jejuni cytotoxicity in the Galleria mellonella infection model. In biologically relevant models, the T6SS enhances C. jejuni interactions with and invasion of chicken primary intestinal cells and enhances the ability of C. jejuni to colonize chickens. This study demonstrates that the C. jejuni T6SS provides defense against oxidative stress and enhances host colonization, and highlights the importance of the T6SS during in vivo survival of T6SS-positive C. jejuni strains.

Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa (intracellular toxicity of S. aureus) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA, encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.

K. pneumoniae sequence type 258 (Kp ST258) is a major cause of healthcare-associated pneumonia. However, it remains unclear how it causes protracted courses of infection in spite of its expression of immunostimulatory lipopolysaccharide, which should activate a brisk inflammatory response and bacterial clearance. We predicted that the metabolic stress induced by the bacteria in the host cells shapes an immune response that tolerates infection. We combined in situ metabolic imaging and transcriptional analyses to demonstrate that Kp ST258 activates host glutaminolysis and fatty acid oxidation. This response creates an oxidant-rich microenvironment conducive to the accumulation of anti-inflammatory myeloid cells. In this setting, metabolically active Kp ST258 elicits a disease-tolerant immune response. The bacteria, in turn, adapt to airway oxidants by upregulating the type VI secretion system, which is highly conserved across ST258 strains worldwide. Thus, much of the global success of Kp ST258 in hospital settings can be explained by the metabolic activity provoked in the host that promotes disease tolerance.