Search

The growing number of sequenced genomes enables the study of secondary metabolite biosynthetic gene clusters (BGC) in phyla beyond well-studied soil bacteria. We mined 2627 enterobacterial genomes to detect 8604 BGCs, including nonribosomal peptide synthetases, siderophores, polyketide-nonribosomal peptide hybrids, and 60 other BGC types, with an average of around 3.3 BGCs per genome. These BGCs represented 212 distinct BGC families, of which only 20 have associated products in the MIBiG standard database with functions such as siderophores, antibiotics, and genotoxins. Pangenome analysis identified genes associated with a specific BGC encoding for colon cancer-related colibactin. In one example, we associated genes involved in the type VI secretion system with the presence of a colibactin BGC inEscherichia. This richness of BGCs in enterobacteria opens up the possibility to discover novel secondary metabolites, their physiological roles and provides a guide to identify and understand PKS associated gene sets.

Type VI Secretion System (T6SS) contributes to both virulence and antimicrobial resistance in Acinetobacter baumannii. Valine-glycine repeat protein G (VgrG) is the core component of T6SS that exists in many bacterial pathogens that have emerged as a potent mediator of pathogenicity in A. baumannii. Two conserved sequences of vgrG 1263-2295 and vgrG1263-1608 were identified antigenic in various strains of Acinetobacter baumannii. The vgrg1263-1608 sequence was implanted in the Loopless C lobe (LCL) from N. meningitidis for surface display and exposure to functional epitopes. The VgrG and LCL-VgrG were expressed and purified. Groups of BALB/c mice were immunized with these proteins and challenged with A. baumannii. Specific IgG titers, whole-cell ELISA, animal survival rates in active and passive immunizations, the bacterial burden in mice tissues, and cytotoxicity of the proteins were determined. The specific IgG suppressed bacterial burdens in the organs, and increased survival rates were noted in the immunized mice. LCL-VgrG immunization provided better protection against A. baumannii infection than the VgrG immunization. The conserved region of VgrG is probably a safe immunogen to effective vaccine development or an antiserum to control A. baumannii infections.

Bacteria have evolved systems dedicated to interbacterial competition. Here we highlight defenses utilized by Gram-negative cells against type VI secretion system (T6SS)-wielding competitors, including physical barriers, genetically encoded antidotes, and stress responses. Further investigation of specific and general defenses will reveal the interbacterial selective pressures impacting bacterial survival in nature.

Clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa from patients with cystic fibrosis (CF) frequently contain mutations in the gene encoding an elongation factor, FusA1. Recent work has shown that fusA1 mutants often display elevated aminoglycoside resistance due to increased expression of the efflux pump, MexXY. However, we wondered whether these mutants might also be affected in other virulence-associated phenotypes. Here, we isolated a spontaneous gentamicin-resistant fusA1 mutant (FusA1(P443L)) in which mexXY expression was increased. Proteomic and transcriptomic analyses revealed that the fusA1 mutant also exhibited discrete changes in the expression of key pathogenicity-associated genes. Most notably, the fusA1 mutant displayed greatly increased expression of the Type III secretion system (T3SS), widely considered to be the most potent virulence factor in the P. aeruginosa arsenal, and also elevated expression of the Type VI (T6) secretion machinery. This was unexpected because expression of the T3SS is usually reciprocally coordinated with T6 secretion system expression. The fusA1 mutant also displayed elevated exopolysaccharide production, dysregulated siderophore production, elevated ribosome synthesis, and transcriptomic signatures indicative of translational stress. Each of these phenotypes (and almost all of the transcriptomic and proteomic changes associated with the fusA1 mutation) were restored to levels comparable with that in the progenitor strain by expression of the WT fusA1 gene in trans, indicating that the mutant gene is recessive. Our data show that in addition to elevating antibiotic resistance through mexXY expression (and also additional contributory resistance mechanisms), mutations in fusA1 can lead to highly selective dysregulation of virulence gene expression.

Avian pathogenic Escherichia coli (APEC) is an important pathogen that causes avian colibacillosis in poultry. APEC infection can lead to pathological changes in chicken trachea. The type VI secretion system (T6SS) of APEC contribute to the pathogenicity of APEC. However, whether T6SS plays a role in infection of the trachea remains unclear. We constructed mutant strain Δhcp2a by the Red recombination method system. The role of hcp2a (the structural secretion components and secretory protein of the T6SS) in the infection of trachea was investigated. The mutation strain displayed a significant increase in biofilm formation and a decrease in resistance to chicken serum. Moreover, RNA sequencing analyses showed that infection of chicken tracheal epithelium by the mutant strain Δhcp2a induced differential expression of genes. The result also showed that 14 genes (13 genes were downregulated) were enriched in cytokine-cytokine receptor interaction signalling pathway at 12 and 24 h post infection. The mutation Δhcp2a resulted in significant decreases in the bacterial loads in trachea at 6 and 12 h post infection. Real-time PCR analyses showed that the hcp2a mutation downregulated the expression of IL8 and IL1β at mRNA level in chicken tracheal epithelium. Our results indicate that mutation of hcp2a influenced genes expression of the cytokine-cytokine receptor interaction pathway by decreasing APEC colonization in the trachea.

The type VI secretion system (T6SS), a multifunctional protein secretion device, plays very important roles in bacterial killing and/or virulence to eukaryotic cells. Although T6SS genes have been found in many Xanthomonas species, the biological function of T6SSs has not been elucidated in most xanthomonads. In this study, we identified two phylogenetically distinct T6SS clusters, T6SS1 and T6SS2, in a newly sequenced Chinese strain GX01 of Xanthomonas oryzea pv. oryzicola (Xoc) which causes bacterial leaf streak (BLS) of rice (Oryza sativa L.). Mutational assays demonstrated that T6SS1 and T6SS2 are not required for the virulence of Xoc GX01 on rice. Nevertheless, we found that T6SS2, but not T6SS1, played an important role in bacterial killing. Transcription and secretion analysis revealed that hcp2 gene is actively expressed and that Hcp2 protein is secreted via T6SS. Moreover, several candidate T6SS effectors were predicted by bioinformatics analysis that might play a role in the antibacterial activity of Xoc. This is the first report to investigate the type VI secretion system in Xanthomonas oryzae. We speculate that Xoc T6SS2 might play an important role in inter-bacterial competition, allowing this plant pathogen to gain niche advantage by killing other bacteria.

Edwardsiella piscicida is an important pathogen that infects a wide range of hosts, from fish to human. Its infection leads to extensive losses in a diverse array of commercially important fish, like Japanese flounder, turbot, and tilapia. During the infection, type III secretion system (T3SS) and type VI secretion system (T6SS) of E. piscicida play significant roles, but how T3SS and T6SS cooperatively contribute to its virulence is still unknown. In this study, we first examined the roles of T3SS and T6SS in different processes during E. piscicida infection of host cells, and revealed that T3SS of E. piscicida is responsible for promoting bacterial invasion, the following intracellular replication and inducing cell death in host cells, while T6SS restrains E. piscicida intracellular replication and cell death in J774A.1 cells, which suggested that T3SS and T6SS antagonistically concert E. piscicida infection. Furthermore, we found an significant decrease in transcription level of IL-1β in zebrafish kidney infected with T3SS mutant and an drastically increase in transcription level of TNF- α infected with T6SS mutant when compared with the wild-type. Interestingly, both T3SS and T6SS mutants showed significant attenuated virulence in the zebrafish infection model when compared with the wild-type. Finally, considering the cooperative role of T3SS and T6SS, we generated a mutant strain WEDΔT6SS based on the existing live attenuated vaccine (LAV) WED which showed improved vaccine safety and comparable immune protection. Therefore, WEDΔT6SS could be used as an optimized LAV in the future. Taken together, this work suggested a bilateral role of T3SS and T6SS which respectively act as spear and shield during E. piscicida infection, together contribute to E. piscicida virulence.

The Type VI secretion system (T6SS) is a membrane protein complex related to inter-bacterial competitions and host-pathogen interactions in Pseudomonas aeruginosa. The T6SS is regulated by a great variety of regulatory mechanisms at multiple levels, including post-translational modification with threonine phosphorylation mediated by Ser/Thr protein kinase PpkA and phosphatase PppA. The T6SS is activated by PpkA via Thr phosphorylation of Fha, and PppA can antagonize PpkA. PppA is a PP2C-family protein phosphatase and plays a key role in the disassembly and reassembly of T6SS organelles. Herein, we report the first crystal structure of PppA from Pseudomonas aeruginosa, which was determined at a resolution of 2.10 A. The overall structure consists of a bacteria PPM structural core and a flexible flap subdomain. PppA harbors a catalytic pocket containing two manganese ions which correspond to the canonical dinuclear metal center of Ser/Thr protein phosphatases including the bacterial PPM phosphatases and human PP2C. The flexibility and the diversity of the sequence of flap subdomain across the homologues might provide clues for substrates specific recognition of phosphatases.

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that is mainly distributed in the seafood such as fish, shrimps and shellfish throughout the world. V. parahaemolyticus can cause diseases in marine aquaculture, leading to huge economic losses to the aquaculture industry. More importantly, it is also the leading cause of seafood-borne diarrheal disease in humans worldwide. With the development of animal model, next-generation sequencing as well as biochemical and cell biological technologies, deeper understanding of the virulence factors and pathogenic mechanisms of V. parahaemolyticus has been gained. As a globally transmitted pathogen, the pathogenicity of V. parahaemolyticus is closely related to a variety of virulence factors. This article comprehensively reviewed the molecular mechanisms of eight types of virulence factors: hemolysin, type III secretion system, type VI secretion system, adhesion factor, iron uptake system, lipopolysaccharide, protease and outer membrane proteins. This review comprehensively summarized our current understanding of the virulence factors in V. parahaemolyticus, which are potentially new targets for the development of therapeutic and preventive strategies.

Glutathione is a low molecular weight thiol that is important for maintaining intracellular redox homeostasis. Some bacteria are able to import exogenous glutathione as a nutritional source and to counter oxidative stress. In cytosolic pathogens Burkholderia pseudomallei and Listeria monocytogenes, host glutathione regulates bacterial virulence. In B. pseudomallei, glutathione activates the membrane-bound histidine kinase sensor VirA that leads to activation of the Type VI Secretion System. In L. monocytogenes, host glutathione leads to the binding of bacterial glutathione to the master virulence regulator PrfA as an allosteric activator. Glutathione can also modulate virulence factors to control their activity by S-glutathionylation. Thus, host glutathione acts as a spacio-temporal cue for some pathogens to switch on their virulence programs at the right time and place.

Type VI secretion system (T6SS), as a macromolecular system, is commonly found in Gram-negative bacteria and responsible for exporting effectors. T6SS consists of 13 core proteins. TssL is a component of the membrane complex and plays a pivotal role in T6SS. Here, we report the crystal structure of the C-terminal periplasmic domain of TssL (TssLCter) from Serratia marcescens FS14. The TssLCter (310-503) contain a five-stranded anti-parallel β-sheet flanked by five α-helices and a short N-terminal helix. Structural comparisons revealed that it belongs to the OmpA-like family with a remarked difference in the conformation of the loop3-5. In OmpA-like family, the corresponding loop is located close to loop2-3, forming a cavity with a small opening together with the longest α5, whereas in TssLCter, loop3-5 flipped away from this cavity region. In addition, significant differences in the peptidoglycan (PG) binding site suggest that big conformational change must take place to accomplish the PG binding for TssLCter. Furthermore, a long flexible loop between helices α1 and α2 is unique in TssL. TssL would have a big conformational change during the delivery of the Hcp needle and effectors. So we speculate that the long flexible endows TssL the adaptation of its evolutionary new function.

Vibrio mimicus distributes widely in the aquatic ecosystem and causes vibriosis in various aquatic animals. For the past few years, Macrobrachium nipponense has been suffering from red body disease with significant economic loss in Jiangsu, and V. mimicus is recognized to be the etiological agent of the disease. In this study we investigated the pathogenicity and antibiotic resistance of V. mimicus Y4 which caused red body disease in M. nipponense in the genomic level. In addition, the high pathogenicity of V. mimicus Y4 was further verified by survival analysis and lesions of hepatopancreas and gill tissues of M. nipponense. The complete genome sequence analysis showed that its genome contained two circular chromosomes and one plasmid with a total size of 4,347,065 bp, which harbored 4084 coding genes. Among these genes, 387 genes were predicted as virulence related genes which could be classified as type IV pilus, flagella, hemolysin, heme receptors, type VI secretion system, and so on, which are involved in the colonization, invasion and destruction of host tissues; 129 genes were predicted as antibiotic resistance genes, including aminoglycoside resistant, macrolide resistant, beta-lactam resistant, and polymyxin resistant genes, which agree with the antibiotic resistance results, which indicated V. mimicus Y4 to be a multidrug-resistant bacterium. The LD50 of the strain Y4 to M. nipponense was 3.28 × 105 CFU/mL at 96 h post-infection. Histopathological analysis exhibited obvious severe lesions of hepatopancreas and gill tissues of diseased M. nipponense, such as the basal laminae of the hepatopancreatic tubules were rupture, vacuolation were decreased, and gill filament cells were broken in diseased prawns. This study provides a theoretical basis for better understanding of the pathogenicity and antibiotic resistance, which might contribute to the prevention of V. mimicus in the future.

The bacterial type VI secretion system (T6SS) delivers effectors into eukaryotic host cells or toxins into bacterial competitor for survival and fitness. The T6SS is positively regulated by the threonine phosphorylation pathway (TPP) and negatively by the T6SS-accessory protein TagF. Here, we studied the mechanisms underlying TagF-mediated T6SS repression in two distinct bacterial pathogens, Agrobacterium tumefaciens and Pseudomonas aeruginosa. We found that in A. tumefaciens, T6SS toxin secretion and T6SS-dependent antibacterial activity are suppressed by a two-domain chimeric protein consisting of TagF and PppA, a putative phosphatase. Remarkably, this TagF domain is sufficient to post-translationally repress the T6SS, and this inhibition is independent of TPP. This repression requires interaction with a cytoplasmic protein, Fha, critical for activating T6SS assembly. In P. aeruginosa, PppA and TagF are two distinct proteins that repress T6SS in a TPP-dependent and -independent pathways, respectively. P. aeruginosa TagF interacts with Fha1, suggesting that formation of this complex represents a conserved TagF-mediated regulatory mechanism. Using TagF variants with substitutions of conserved amino acid residues at predicted protein-protein interaction interfaces, we uncovered evidence that the TagF-Fha interaction is critical for TagF-mediated T6SS repression in both bacteria. TagF inhibits T6SS without affecting T6SS protein abundance in A. tumefaciens, but TagF overexpression reduces the protein levels of all analyzed T6SS components in P. aeruginosa. Our results indicate that TagF interacts with Fha, which in turn could impact different stages of T6SS assembly in different bacteria, possibly reflecting an evolutionary divergence in T6SS control.

Summary As a common gut pathogen, Campylobacter jejuni (C. jejuni) harbors the Type VI Secretion System (T6SS) that injects toxic effectors into neighboring cells, modulating microbial competitions in the harsh gut environment. Using bile salt as a natural stressor and T6SS-positive C. jejuni as a predator, we show that T6SS activity could entail a cost during bacterial predation under environmental stress. Our data suggest bile salt influx and subsequent DNA damage due to the prey-driven activation of the T6SS. We further combined experiments and mathematical modeling to explore how the stress-induced “predation cost” determines ecological outcomes. Consistent with a population-dynamics model, we found predator extinction above a critical bile salt concentration and prey-predator coexistence below this level. Moreover, we utilized the predation cost as an effective strategy facilitating host defense against C. jejuni infection. Together, we elucidate how predator dominance versus extinction emerges from the interplay between environmental stress and the T6SS machinery., Graphical abstract, Highlights • Campylobacter jejuni uses Type VI secretion system (T6SS) to kill prey bacteria • Under bile salt stress, activated T6SS may promote bile salt uptake and DNA damage • T6SS-dependent predation by C. jejuni thus entails a “predation cost” under stress • The predation cost leads to predator extinction and host defense against C. jejuni, Experimental models in systems biology; Microbiology

The aim of this study was to isolates with antagonist activity against R. solanacearum. Thirty-two bacterial isolates were obtained from samples, and they were screened for potential antagonistic activity against R. Solanacearum. Using the agar spot method, ten out of the 21 tested bacteria showed antilisterial activity. VIH2 had the highest inhibitory effect on the growth of R. Solanacearum. Based on 16S rDNA and Biolog test analysis, the strain VIH2 was identified as Pseudomonas aeruginosa. Single-factor and Response Surface Methodology experiments were used to optimize the culture medium and conditions. This study was to explore whether the hemolysin-co-regulated protein secretion island I (HSI-I)-encoded type VI secretion system (T6SS) in Pseudomonas can be used as a biological control approach against Ralstonia solanacearum under field conditions. Bacterial competition assay showed that the HSI-I type T6SS of strain VIH2 exhibited dramatic antibacterial killing activity against R. solanacearum. The HSI-I T6SS of P. aeruginosa was regulated by the ppKA gene. We disrupted the gene ppKA in VIH2 by a single crossover to yield the VIH2 (ΔppKA) mutant. The antagonism of VIH2 was significantly decreased by ppKA gene disruption. In conclusion, our data supported the idea that HSI-I T6SS plays a crucial role in the antagonistic action of strain VIH2 against R. solanacearum. This alternative approach for antagonism against R. solanacearum might help develop attenuated strains of engineered bacteria for biological control.

Using physical force to translocate macromolecules across a membrane has the advantage of being a universal solution independent of the properties of the target membrane. However, physically punching a stiff membrane is not a trivial task and three things are necessary for success: a sharp tip, a source of energy, and the ability to strongly bind to the target. In this review we describe the basic mechanism of membrane puncturing by contractile nanomachines with a focus on the T4 phage, R-type pyocin, and the bacterial Type VI secretion system (T6SS) based on recent studies of the structures and dynamics of their assembly.

Specialized secretion systems are infamous for their contribution to host–pathogen interactions. Our discovery that the type VI secretion system delivers toxins between bacterial cells has broadened our understanding of how both pathogens and non-pathogens interact with one another, whether within or outside of the host.

The intracellular bacterial pathogen Francisella tularensis causes tularemia, a zoonosis that can be fatal. The type VI secretion system (T6SS) encoded by the Francisella pathogenicity island (FPI) is critical for the virulence of this organism. Existing studies suggest that the complete repertoire of T6SS effectors delivered to host cells is encoded by the FPI. Using a proteome-wide approach, we discovered that the FPI-encoded T6SS exports at least three effectors encoded outside of the island. These proteins share features with virulence determinants of other pathogens, and we provide evidence that they can contribute to intramacrophage growth. The remaining proteins that we identified are encoded within the FPI. Two of these FPI-encoded proteins constitute effectors, whereas the others form a unique complex required for core function of the T6SS apparatus. The discovery of secreted effectors mediating interactions between Francisella and its host significantly advances our understanding of the pathogenesis of this organism.

The human pathogen Pseudomonas aeruginosa harbors three paralogous zinc proteases annotated as AmpD, AmpDh2, and AmpDh3, which turn over the cell wall and cell-wall-derived muropeptides. AmpD is cytoplasmic and plays a role in recycling of cell-wall muropeptides, with a link to antibiotic resistance. AmpDh2 and AmpDh3 are periplasmic enzymes; with the former anchored to the inner leaflet of the outer membrane and the latter a soluble enzyme. We document herein that the type VI secretion system locus II (H2-T6SS) of P. aeruginosa delivers AmpDh3 (but not AmpD or AmpDh2) to the periplasm of a prey bacterium upon contact. AmpDh3 hydrolyzes the cell-wall peptidoglycan of the prey bacterium, which leads to its killing, thereby providing a growth advantage for P. aerugionsa in bacterial competition. We also document that the periplasmic protein PA0808, heretofore of unknown function, affords self-protection from lysis by AmpDh3. Cognates of the AmpDh3-PA0808 pair are widely distributed across Gram-negative bacteria, underscoring the importance of their function as an evolutionary advantage and that of the H2-T6SS as the means for manifestation of the effect.

Signal transduction system and specialized secretory devices are crucial for bacteria to sense and adequately adapt in adverse environmental conditions. Therefore, it's crucial for microbes to detect and respond to lethal attacks when envelope is perturbed so as to minimize and fix the damage in milieu. We investigated the adaptive response of porcine extra-intestinal pathogenic Escherichia coli PCN033 to polymyxin B challenge. Treatment with polymyxin B led to rapid and robust activation of Rcs system via RcsF, as well as the accumulation of reactive oxygen species. ExPEC T6SS expression was strongly induced by RcsB in Rcs system, resulting in the reduction in the damage to constitute a survival strategy. Finally, we show that T6SS of ExPEC is involved in its pathogenicity in mouse model. Compared with the wild type strain, the deletion of T6SS genes led to a decrease in the organ colonization ability, and the RcsFS2DM3Q mutant that caused Rcs activation had a stronger colonization ability than the wild type strain. In conclusion, Rcs system orchestrates Rcs cascade to trigger antioxidant defense of T6SS, and presents a typical model in which a bacterium reschedule its transcription network via the Rcs phosphorelay pathway in response to membrane perturbations for survival and pathogenesis.

Vibrio parahaemolyticus (VP) is a major pathogen that causes acute hepatopancreatic necrosis disease (AHPND) in shrimp. Among the multiple pathogenic factors, the type VI secretion system (T6SS) plays an important role in pathogenesis caused by VP. In the present study, we examined the effective bactericidal concentrations of ethanol-extracts and water-extracts of Sanguisorba officinalic L. (S. officinalic) on VPAHPND, a causative agent of AHPND. Moreover, we also investigated the dose-dependent effects of these extracts on the growth of VPAHPND and the expression levels of seven T6SS-related genes, including VP1388, VP1393, VP1400, VP1409, VPA1027, VPA1043 and VPA104. The results showed that although both extracts could inhibit the growth of VPAHPND, the ethanol-extract possessed the stronger antibiotic effect and could kill VPAHPND at 6.4 mg/mL within 10 h. Analysis of the expression levels of T6SS-related genes indicated that ethanol-extract at higher dosages could up-regulate the expression of the T6SS1 gene within a short period of time, although such effect was attenuated with time. In addition, the ethanol-extract could significantly inhibit the expression of T6SS2 at the mRNA level in a dose-dependent manner. Based on the function of each gene, we speculated that the ethanol-extract could weaken the adhesion of VPAHPND, reduce its infectivity to the host, and block the activation of other T6SS-related virulence factors through inhibiting the initiation of T6SS, leading to the attenuated pathogenicity of VPAHPND to the host. The results of a clinical trial confirmed that dried garden burnet had a strong inhibitory effect on Vibrio proliferation in shrimp. Taken together, our results confirmed that S. officinalic could be used as a potentially effective agent for controlling and even preventing VPAHPND infection.

Motile aeromonad septicemia (MAS) caused by Aeromonas hydrophila is one of the common bacterial causes of fish mortalities. Prophylactic vaccination against this and other diseases is essential for continued growth of aquaculture. The type VI secretion system (T6SS) plays a crucial role in the virulence of A. hydrophila. The hemolysin co-regulated protein (Hcp) is an integral component of the T6SS apparatus and is considered a hallmark of T6SS function. Here, the T6SS effector Hcp was expressed and characterized, and its immunogenicity and protective efficacy were evaluated in common carp (Cyprinus carpio). Hcp secretion was found to be strongly induced by low temperature in A. hydrophila. Immunoblot analysis demonstrated that Hcp is conserved among A. hydrophila strains of different origins. The vaccination with recombinant Hcp resulted in an increased survival (46.67%) in common carp during a 10-day challenge time compared to non-vaccinated fish (7.14%). The vaccinated fish also showed the significantly increased levels of IgM antibody in serum and cytokines such as inerleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in kidney, spleen and gills. The recombinant Hcp shows promise as a vaccine candidate against A. hydrophila.

Bacteria use the type VI secretion system (T6SS) to kill neighboring cells. One key feature of the T6SS is the secretion of diverse effectors. Here, we discuss six publications that describe three superfamilies of T6SS proteins, each dedicated to mediate the secretion of cognate effectors.

Type VI secretion systems (T6SSs) are nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized a Salmonella T6SS antibacterial effector called Tlde1 that is toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tldi1). Microscopy analysis reveals that cells expressing Tlde1 stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovers similarities between Tlde1 and the catalytic domain of l,d-transpeptidases. Point mutations on conserved catalytic residues abrogate toxicity. Biochemical assays reveal that Tlde1 displays both l,d-carboxypeptidase activity by cleaving peptidoglycan tetrapeptides between meso-diaminopimelic acid3 and d-alanine4 and l,d-transpeptidase exchange activity by replacing d-alanine4 by a non-canonical d-amino acid. Phylogenetic analysis shows that Tlde1 homologs constitute a family of T6SS-associated effectors broadly distributed among Proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition and reveals a different mechanism of bacterial antagonism.

Francisella bacteria rely on a phylogenetically distinct Type VI Secretion System (T6SS) to escape host phagosomes and cause the fatal disease tularemia, but the structural and molecular mechanisms involved are unknown. Here, we report the atomic structure of the Francisella T6SS central spike complex, obtained by cryo-electron microscopy. Our structural and functional studies demonstrate that, unlike the single-protein spike composition of other T6SS subtypes, Francisella T6SS’s central spike is formed by two proteins, PdpA and VgrG, akin to T4-bacteriophage gp27 and gp5, respectively, and that PdpA has unique characteristics, including a putative cargo within its cavity and an N-terminal helical lid. Structure-guided mutagenesis demonstrates that the PdpA N-terminal lid and C-terminal spike are essential to Francisella T6SS function. PdpA is thus both an adaptor, connecting VgrG to the tube, and a likely carrier of secreted cargo. These findings are important to understanding Francisella pathogenicity and designing therapeutics to combat tularemia.

Campylobacter jejuni is a major cause of infectious diarrhea in humans. The bacterium can be transmitted through contaminated poultry meat and waste water. We report the presence of C. jejuni from potential transmission sources including egg shells, poultry waste, waste water and migratory bird droppings with a prevalence rate of 78%, 66%, 86% and 70% respectively. Antibiotic resistance profile showed high number of isolates resistant to multiple antibiotics including 4th generation cephalosporins. C. jejuni isolates were further screened for presence of T6SS, an important virulence factor. None of the C. jejuni isolates from migratory birds carried a T6SS, whereas highest prevalence of T6SS isolates was observed in waste water samples, followed by poultry waste and egg shells. To determine virulence potential of the isolates, hemolytic activity of isolates was compared. Although variation in hemolytic potential between isolates from different sources was noted, higher hemolytic activity was observed for isolates possessing hcp, a T6SS gene. Furthermore, presence of T6SS affords the bacterium some survival advantage when compared to T6SS competent Helicobacter pullorum which occupies the same niche. Taken together our findings indicate that C. jejuni with T6SS have a fitness advantage increasing their isolation frequency from waste water and poultry waste.

Summary 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., Graphical Abstract, Highlights • The structure of the VgrG2b C-terminal domain presents a metallopeptidase fold • VgrG2b exerts antibacterial activity in the periplasmic space • Toxicity of VgrG2b is counteracted by a cognate periplasmic immunity protein • VgrG2bC-ter-intoxicated prey cells bleb at the midcell and lyse, The bacterial type VI secretion system (T6SS) delivers effector proteins into prokaryotic and eukaryotic cells to enhance the survival of the donor cell. Wood et al. describe an antibacterial T6SS toxin family eliciting a profound cell division defect and lysis. The structure of this periplasmic-acting toxin reveals a metallopeptidase fold.

The type VI secretion system (T6SS) delivers toxic effectors between Gram-negative bacteria. Most antibacterial T6SS effectors are peptidoglycanases, nucleases, or lipases. In the current work, Tang et al. structurally and functionally characterize a novel family of NAD(P)+-hydrolyzing effectors (NADases), thus expanding the documented types of T6SS substrates. Bioinformatic identification of NADase family members putatively secreted by the bacteriolytic type VII secretion system (T7SS) of Gram-positive bacteria further points to NADases as a diverse and important class of effectors.

The type VI secretion system, an organelle common to Gram-negative bacteria, is disassembled in a process mediated by the binding of ClpV ATPase. Fluorescence microscopy experiments in Vibrio cholerae have revealed that, during the course of this disassembly, the organelle breaks into pieces which appear to contract. We developed and analysed a dynamic model of ClpV binding. Our findings suggest that this breaking behavior is not directly triggered by accumulated ClpV binding.

Summary Analysis of genes required for host infection will provide clues to the drivers of evolutionary fitness of pathogens like Vibrio cholerae , a mounting threat to global heath. We used transposon insertion site sequencing (Tn-seq) to comprehensively assess the contribution of nearly all V. cholerae genes toward growth in the infant rabbit intestine. Four hundred genes were identified as critical to V. cholerae in vivo fitness. These included most known colonization factors and several new genes affecting the bacterium's metabolic properties, resistance to bile, and ability to synthesize cyclic AMP-GMP. Notably, a mutant carrying an insertion in tsiV3 , encoding immunity to a bacteriocidal type VI secretion system (T6SS) effector VgrG3, exhibited a colonization defect. The reduced in vivo fitness of tsiV3 mutants depends on their cocolonization with bacterial cells carrying an intact T6SS locus and VgrG3 gene, suggesting that the V. cholerae T6SS is functional and mediates antagonistic interbacterial interactions during infection.

The warfare among microbial species as well as between pathogens and hosts is fierce, complicated, and continuous. In Pseudomonas aeruginosa, the muramidase effector Tse3 (Type VI secretion exported 3) can be injected into the periplasm of neighboring bacterial competitors by a Type VI secretion apparatus, eventually leading to cell lysis and death. However, P. aeruginosa protects itself from lysis by expressing immune protein Tsi3 (Type six secretion immunity 3). Here, we report the crystal structure of the Tse3-Tsi3 complex at 1.8 Å resolution, revealing that Tse3 possesses one open accessible, goose-type lysozyme-like domain with peptidoglycan hydrolysis activity. Calcium ions bind specifically in the Tse3 active site and are identified to be crucial for its bacteriolytic activity. In combination with biochemical studies, the structural basis of self-protection mechanism of Tsi3 is also elucidated, thus providing an understanding and new insights into the effectors of Type VI secretion system.

SUMMARY Secretion systems require high-fidelity mechanisms to discriminate substrates among the vast cytoplasmic pool of proteins. Factors mediating substrate recognition by the type VI secretion system (T6SS) of Gram-negative bacteria, a widespread pathway that translocates effector proteins into target bacterial cells, have not been defined. We report that haemolysin coregulated protein (Hcp), a ring-shaped hexamer secreted by all characterized T6SSs, binds specifically to cognate effector molecules. Electron microscopy analysis of an Hcpeffector complex from Pseudomonas aeruginosa revealed the effector bound to the inner surface of Hcp. Further studies demonstrated that interaction with the Hcp pore is a general requirement for secretion of diverse effectors encompassing several enzymatic classes. Though previous models depict Hcp as a static conduit, our data indicate it is a chaperone and receptor of substrates. These unique functions of a secreted protein highlight fundamental differences between the export mechanism of T6 and other characterized secretory pathways.

International audience; The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly.

The Serratia entomophila antifeeding prophage (Afp) is a bullet-shaped toxin-delivery apparatus similar to the R-pyocins of Pseudomonas aeruginosa. Morphologically it resembles the sheathed tail of bacteriophages such as T4, including a baseplate at one end. It also shares features with the type VI secretion systems. Cryo-electron micrographs of tilted Afp specimens (up to 60 degrees) were analyzed to determine the correct cyclic symmetry to overcome the limitation imposed by exclusively side views in nominally untilted specimens. An asymmetric reconstruction shows clear 6-fold cyclic symmetry contrary to a previous conclusion of 4-fold symmetry based on analysis of only the preferred side views (Sen, A., Rybakova, D., Hurst, M. R., and Mitra, A. K. (2010) J. Bacteriol. 192, 4522-4525). Electron tomography of negatively stained Afp revealed right-handed helical striations in many of the particles, establishing the correct hand. Higher quality micrographs of untilted specimens were processed to produce a reconstruction at 2.0-nm resolution with imposed 6-fold symmetry. The helical parameters of the sheath were determined to be 8.14 nm for the subunit rise along and 40.5° for the rotation angle around the helix. The sheath is similar to that of the T4 phage tail but with a different arrangement of the subdomain of the polymerizing sheath protein(s). The central tube is similar to the diameter and axial width of the Hcp1 hexamer of P. aeruginosa type VI secretion system. The tube extends through the baseplate into a needle resembling the "puncture device" of the T4 tail. The tube contains density that may be the toxin and/or a length-determining protein.

The Type VI secretion system (T6SS) is the most recently described of the Gram-negative bacterial secretion systems and is widely distributed amongst diverse species. T6SSs are currently believed to be complex molecular machines which inject effector proteins into target cells and which incorporate a bacteriophage-like cell-puncturing device. T6SSs have been implicated in eukaryotic cell targeting and virulence in a range of important pathogens. More recently, 'antibacterial' T6SSs have been reported, which are used to efficiently target competitor bacterial cells by the injection of antibacterial toxins. Although it is clear that T6SSs can be deployed as versatile weapons to compete with other bacteria or attack simple or higher eukaryotes, much remains to be determined about this intriguing system.

The type VI secretion system (T6SS) of Gram-negative bacteria has been implicated in microbial competition; however, which components serve purely structural roles, and which serve as toxic effectors remains unresolved. Here, we present evidence that VgrG-3 of the Vibrio cholerae T6SS has both structural and toxin activity. Specifically, we demonstrate that the C-terminal extension of VgrG-3 acts to degrade peptidoglycan and hypothesize that this assists in the delivery of accessory T6SS toxins of V. cholerae. To avoid self-intoxication, V. cholerae expresses an anti-toxin encoded immediately downstream of vgrG-3 that inhibits VgrG-3-mediated lysis through direct interaction.

Vibrio alginolyticus, a Gram-negative marine bacterium, has brought about severe economic damage to the mariculture industry by causing vibriosis in various fish species. We are intrigued in the regulation of the pathogenesis in this bacterium. Here, we reported a complex regulatory connection among the newly defined type VI secretion system (T6SS), quorum sensing (QS), and 3',5'-cyclic diguanylic acid (c-di-GMP) signal through the phosphatase PppA encoded in the T6SS gene cluster of V. alginolyticus. Whole-genome transcriptome analysis revealed various regulatory targets of PppA including the T6SS substrate hemolysin coregulated protein (Hcp), quorum sensing regulator LuxR, exotoxin alkaline serine protease (Asp), flagellar proteins, as well as proteins involved in polysaccharide biosynthesis and transport. Western blot analysis showed PppA served as a negative regulator of the expression and secretion of Hcp1. Mutation of pppA resulted in an increased level of the intracellular second messenger c-di-GMP and a decreased expression of the QS regulator LuxR as well as exotoxin Asp. Complementation of intact pppA gene in ΔpppA mutant restored the production of c-di-GMP, LuxR, and Asp to the wild-type level. Phenotypic studies suggested that PppA takes part in the modulation of biofilm formation, motility, and cell aggregation. These results demonstrated new roles of PppA in controlling virulence factors and pleiotropic phenotypes and contributed to our understanding of the regulation of pathogenesis in V. alginolyticus.

Among specialized bacterial secretion systems, the most widespread is the type VI secretion system (T6SS). This transports effector molecules into target cells in a single, cell-contact dependent step. T6SSs are structurally related to the cell-puncturing device of tailed bacteriophages and predicted to function as contractile injection machineries that perforate eukaryotic and prokaryotic target membranes for effector delivery. Activities of T6SSs can play important roles in virulence by modifying the eukaryotic host cytoskeleton through actin crosslinking. They are also efficient weaponry in interbacterial warfare and provide a fitness advantage by hydrolyzing cell walls of opponent bacteria. The role of T6SSs in interbacterial competition might enable pathogens to outcompete commensal bacteria and facilitate host colonization.

Recently, it was identified that Pseudomonas aeruginosa competes with rival cells to gain a growth advantage using a novel mechanism that includes two interrelated processes as follows: employing type VI secretion system (T6SS) virulence effectors to lyse other bacteria, and at the same time producing specialized immunity proteins to inactivate their cognate effectors for self-protection against mutual toxicity. To explore the structural basis of these processes in the context of functional performance, the crystal structures of the T6SS virulence effector Tse1 and its complex with the corresponding immunity protein Tsi1 were determined, which, in association with mutagenesis and Biacore analyses, provided a molecular platform to resolve the relevant structural questions. The results indicated that Tse1 features a papain-like structure and conserved catalytic site with distinct substrate-binding sites to hydrolyze its murein peptide substrate. The immunity protein Tsi1 interacts with Tse1 via a unique interactive recognition mode to shield Tse1 from its physiological substrate. These findings reveal both the structural mechanisms for bacteriolysis and the self-protection against the T6SS effector Tse1. These mechanisms are significant not only by contributing to a novel understanding of niche competition among bacteria but also in providing a structural basis for antibacterial agent design and the development of new strategies to fight P. aeruginosa.

Edwardsiella tarda is a Gram-negative broad-host-range pathogen that causes hemorrhagic septicemia in many commercially important fish species. Its ability to adapt to and thrive in diverse environments outside and inside of its hosts prompts us to investigate the roles of the previously identified 33 putative two-component signal transduction systems (TCSs) in E. tarda. In this work, we successfully constructed deletion mutations in each of the response regulator genes, suggesting that none of the TCSs are essential for cell viability in E. tarda. The mutants were further examined for roles in biofilm formation, antibiotic resistance, stress response, expression and secretion of proteins involved in either the type III secretion system (T3SS) or type VI secretion system (T6SS), as well as virulence. Through these assays, we identified four regulators of biofilm development, two regulators of antibiotic resistance, and four regulators involved in stress responses. We found that two regulators, EsrB and PhoP, are essential for the pathogenicity of E. tarda and further demonstrated that these two regulators have codependent and independent contributions to E. tarda virulence. Mutation of EsrB resulted in the complete loss of both the T3SS and T6SS proteins, while PhoP partially regulated the expression of T3SS and T6SS genes through EsrB, and was essential for resistance to antimicrobial peptides. This work suggested that these two response regulators are involved in the regulation of the complex virulence network of this bacterium and merit as candidate genes for live attenuated vaccine construction.

Background: The IcmF family protein TssM is a conserved T6SS component. Results: TssM exhibits ATPase activity, and its impaired ATP binding/hydrolysis activity causes loss of TssM-TssL-Hcp complex formation and Hcp secretion. Conclusion: TssM functions as a T6SS energizer to recruit Hcp into the TssM-TssL complex and powers Hcp secretion. Significance: This is the first demonstration of TssM ATPase activity and its role in protein secretion., The type VI secretion system (T6SS) with diversified functions is widely distributed in pathogenic Proteobacteria. The IcmF (intracellular multiplication protein F) family protein TssM is a conserved T6SS inner membrane protein. Despite the conservation of its Walker A nucleotide-binding motif, the NTPase activity of TssM and its role in T6SS remain obscure. In this study, we characterized TssM in the plant pathogen Agrobacterium tumefaciens and provided the first biochemical evidence for TssM exhibiting ATPase activity to power the secretion of the T6SS hallmark protein, hemolysin-coregulated protein (Hcp). Amino acid substitutions in the Walker A motif of TssM caused reduced ATP binding and hydrolysis activity. Importantly, we discovered the Walker B motif of TssM and demonstrated that it is critical for ATP hydrolysis activity. Protein-protein interaction studies and protease susceptibility assays indicated that TssM undergoes an ATP binding-induced conformational change and that subsequent ATP hydrolysis is crucial for recruiting Hcp to interact with the periplasmic domain of the TssM-interacting protein TssL (an IcmH/DotU family protein) into a ternary complex and mediating Hcp secretion. Our findings strongly argue that TssM functions as a T6SS energizer to recruit Hcp into the TssM-TssL inner membrane complex prior to Hcp secretion across the outer membrane.

Pathogenic microorganisms have to face hostile environments while colonizing and infecting their hosts. Unfortunately, they can cope with it and have evolved a number of complex secretion systems, which direct virulence factors either at the bacterial cell surface into the environmental extracellular milieu or into the host cell cytosol. Six different classes of secretion systems have been described so far, currently identified as type I secretion system (T1SS) up to type VI secretion system (T6SS). The Gram-negative opportunistic human pathogen Pseudomonas aeruginosa possesses a broad panel of secretion systems. Five of the six secretion machines characterized in Gram-negative bacteria are at P. aeruginosa disposal, sometimes in several copies. All these machines are dedicated to the specific secretion of exoproteins, which display various activities useful for bacterial adaptation to the environment or for bacterial pathogenicity. This review will summarize the functional organization of these different secretion systems, which could constitute potential targets for therapeutic treatment of patients infected by one of the most potent nosocomial pathogens identified nowadays.

SummaryThe functional spectrum of a secretion system is defined by its substrates. Here we analyzed the secretomes of Pseudomonas aeruginosa mutants altered in regulation of the Hcp Secretion Island-I-encoded type VI secretion system (H1-T6SS). We identified three substrates of this system, proteins Tse1–3 (type six exported 1–3), which are coregulated with the secretory apparatus and secreted under tight posttranslational control. The Tse2 protein was found to be the toxin component of a toxin-immunity system and to arrest the growth of prokaryotic and eukaryotic cells when expressed intracellularly. In contrast, secreted Tse2 had no effect on eukaryotic cells; however, it provided a major growth advantage for P. aeruginosa strains, relative to those lacking immunity, in a manner dependent on cell contact and the H1-T6SS. This demonstration that the T6SS targets a toxin to bacteria helps reconcile the structural and evolutionary relationship between the T6SS and the bacteriophage tail and spike.

Type VI secretion systems (T6SSs) have been identified recently in several Gram-negative organisms and have been shown to be associated with virulence in some bacterial pathogens. A T6SS of Yersinia pestis CO92 (locus YPO0499-YPO0516) was deleted followed by investigation of the phenotype of this mutation. We observed that this T6SS locus of Y. pestis was preferentially expressed at 26 degrees C in comparison to 37 degrees C suggesting a possible role in the flea cycle. However, we found that the deletion of T6SS locus YPO0499-YPO0516 in Y. pestis CO92 had no effect on the ability of this strain to infect the oriental rat flea, Xenopsylla cheopis. Nevertheless, this mutant displayed increased intracellular numbers in macrophage-like J774.A1 cells after 20 h post-infection for bacterial cells pre-grown at 26 degrees C indicating that expression of this T6SS locus limited intracellular replication in macrophages. In addition, deletion of the YPO0499-YPO0516 locus reduced the uptake by macrophages of the Y. pestis mutant pre-grown at 37 degrees C, suggesting that this T6SS locus has phagocytosis-promoting activity. Further study of the virulence of the T6SS mutant in murine bubonic and inhalation plague models revealed no attenuation in comparison with the parental CO92 strain.

Edwardsiella tarda is a gram-negative pathogen for hemorrhagic septicemia in a broad range of hosts. The type VI secretion system (T6SS) has recently been dissected in E. tarda to secrete EvpC, EvpI and a novel effector protein EvpP. In this study, sequencing and genetic alignments showed that evpP genes from different E. tarda isolates were highly similar and an evpP homolog was also found in Aeromonas hydrophila 0865 isolated from a diseased eel, suggesting the possible lateral gene transfer of evpP or the whole T6SS gene island. With reporter strains carrying gfp gene fused to the evpP promoter region, flow cytometric analysis revealed that transcription of evpP was positively regulated by either the two-component system EsrA-EsrB in E. tarda or the iron concentration in media. Compared with the parental strain, in-frame deletion of evpP in E. tarda EIB202 led to the significantly increased 50% lethal doses in zebrafish (Danio rerio) and Japanese flounder (Paralichthys olivaceus), decreased hemolytic activities, failure to adhere to mucus and reduced serum resistance, and complementation of an intact evpP gene restored these phenotypes in the evpP mutant. Investigation of infection kinetics indicated that the evpP deletion mutant was unable to proliferate in vivo, particularly in immune organs of fish. Moreover, the evpP deletion mutant exhibited incapacity to internalize in EPC cell model in vitro, demonstrating that EvpP in T6SS plays critical roles for invasion mechanism of E. tarda and merits as potential target for attenuated live vaccine construction.

The Type Six Secretion System (T6SS) is a bacterial toxin-delivery system targeting bacterial cells which neighbor the donor, promoting recipient cell death. The T6SS is widely conserved among Gram-negative bacteria and may be a central determinant in bacterial fitness in polymicrobial communities of particular relevance to chronic infection. Sequence homology of secretion system components to the T4 bacteriophage tail spike, cryoEM reconstructions of the secretion system and fluorescence imaging are all consistent with a dynamic mechanism of secretion. The complex system, which is composed of at least 15 proteins, forms a puncturing apparatus/delivery system which uses a donor protein filament to puncture the recipient cell wall to deliver protein toxins. Using quantitative imaging analysis of multiple fluorescent fusions, we present a detailed characterization of T6SS system dynamics visualized in single cells in multiple bacterial species, developing a model of T6SS function. We present quantitative measurements of the dynamics of the secretion system - from the assembly to contraction to disassembly - in conjunction with quantitative measures of system function, including recipient cell lysis.

Bacterial type VI secretion system (T6SS) is a nanomachine that works similarly to a speargun. Rapid contraction of a sling (sheath) drives a long shaft (Hcp) with a sharp tip and associated effectors through the target cell membrane. We show that the amount and composition of the tip regulates initiation of full-length sheath assembly and low amount of available Hcp decreases sheath length. Importantly, we show that both tip and Hcp are exchanged by T6SS among by-standing cells within minutes of initial cell-cell contact. The translocated proteins are reused for new T6SS assemblies suggesting that tip and Hcp reach the cytosol of target cells. The efficiency of protein translocation depends on precise aiming of T6SS at the target cells. This interbacterial protein complementation can support T6SS activity in sister cells with blocked protein synthesis and also allows cooperation between strains to increase their potential to kill competition. VIDEO ABSTRACT.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC), a devastating disease causing millions of dollars production losses and treatment costs. E. ictaluri has variety of distinct mechanisms to survive in the stressful host environment. Type VI Secretion System (T6SS) is involved in survival of intracellular bacteria inside the host immune cells. However, the role of T6SS in E. ictaluri virulence is not known yet. Thus, the purpose of this research was to understand the extent to which T6SS is involved in E. ictaluri virulence. To this aim, we generated an E. ictaluri T6SS mutant (EiΔevpF) and assessed its virulence in channel catfish. The fish trials indicated that EiΔevpF is highly attenuated in channel catfish. Also, vaccination of catfish using the EiΔevpF mutant provided a good protection against wild-type E. ictaluri infection in catfish.

Summary 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., Graphical Abstract, Highlights • A type VI secretion DNase effector (Tde) family identified in Agrobacterium tumefaciens • Tde exhibits antibacterial DNase activity and is counteracted by immunity protein Tdi • Tde confers a competitive advantage to A. tumefaciens during host colonization • Tde and Tdi superfamilies are broadly conserved in bacteria, Type VI secretion systems (T6SSs) are weapons involved in interbacterial competition. Ma et al. report the discovery of a class of T6SS-dependent DNases that support the survival and fitness of the plant pathogen Agrobacterium tumefaciens against competitors such as Pseudomonas aeruginosa during plant colonization.

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.