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Assembly of the Type VI secretion system. Schematic representation of the different stages of T6SS biogenesis. The TssJ-L-M membrane complex is first assembled in the cell envelope (IM, inner membrane; PG, peptidoglycan; OM, outer membrane) and recruits the baseplate-like assembly platform constituted of TssE, TssF, TssG, TssK, VgrG and possibly TssA (platform represented as a green disk with the VgrG green screw). Polymerization of the tail tube (Hcp rings, black rectangles) and sheath (TssBC strands, red rectangles) is initiated after completion of the platform.
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The Type VI secretion system (T6SS) is a widespread weapon dedicated to the delivery of toxin proteins into eukaryotic and prokaryotic cells. The 13 T6SS subunits assemble a cyto-plasmic contractile structure anchored to the cell envelope by a membrane-spanning complex. This structure is evolutionarily, structurally and functionally related to the...
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Context 1
... estingly, contacts with TssK and VgrG require the pre-formation of the TssF-G complex. Taken together these data support the idea that the T6SS baseplate is composed of the VgrG, TssE, TssF, TssG and TssK subunits, on which the Hcp tube and the TssB-TssC sheath will sequentially polymerize (Fig 7). Indeed, co-localization studies showed that TssF is recruited to the apparatus prior to sheath extension. ...
Context 2
... T6SS biogenesis, the results presented here as well as three recent studies [15,40,46] support the proposal that the TssLMJ membrane complex is first assembled and that the T6SS is built by the hierarchical addition of TssK and TssFG. Alternatively, complete baseplates may assemble prior to docking to the membrane complex (Fig 7). Indeed, the baseplate-like structure is anchored to the inner membrane by a network of interactions including contacts between TssK and TssL, TssK and TssM, and TssG and TssM ([40] and this study). ...
Context 3
... how a 6-fold symmetry structure successfully and func- tionally associates to a 5-fold symmetry docking station remains to be elucidated. Once the baseplate is docked to the TssJLM complex, the Hcp inner tube/TssBC outer sheath polymeri- zation can proceed (Fig 7). Indeed, interaction studies showed that TssF and TssG are con- nected to Hcp and TssC, and might initiate tail extension. ...
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Citations
... The biogenesis, contraction, and disassembly of T6SS can be outlined in the following steps: (i) the membrane complex is first embedded in the membrane and recruits the baseplate along with the VgrG tip. Subsequently, the Hcp tube and the TssB/C sheath polymerize and extend from the baseplate; (ii) sheath contraction facilitates the injection of the VgrG tip, Hcp tube, and other effector proteins into neighboring cells or extracellu lar environments; and (iii) ClpV recycles the sheath components for the next rounds of assembly (9)(10)(11). ...
Type VI secretion system (T6SS) is a potent weapon employed by various Pseudomonas species to compete with neighboring microorganisms for limited nutrients and ecological niches. However, the involvement of T6SS effectors in interbacterial competition within the phytopathogen Pseudomonas syringae remains unknown. In this study, we examined two T6SS clusters in a wild-type P. syringae MB03 and verified the involvement of one cluster, namely, T6SS-1, in interbacterial competition. Additionally, our results showed that two T6SS DNase effectors, specifically Tde1 and Tde4, effectively outcompeted antagonistic bacteria, with Tde4 playing a prominent role. Furthermore, we found several cognate immunity proteins, including Tde1ia, Tde1ib, and Tde4i, which are located in the downstream loci of their corresponding effector protein genes and worked synergistically to protect MB03 cells from self-intoxication. Moreover, expression of either Tde1 or C-terminus of Tde4 in Escherichia coli cells induced DNA degradation and changes in cell morphology. Thus, our results provide new insights into the role of the T6SS effectors of P. syringae in the interbacterial competition in the natural environment.
IMPORTANCE
The phytopathogen Pseudomonas syringae employs an active type VI secretion system (T6SS) to outcompete other microorganisms in the natural environment, particularly during the epiphytic growth in the phyllosphere. By examining two T6SS clusters in P. syringae MB03, T6SS-1 is found to be effective in killing Escherichia coli cells. We highlight the excellent antibacterial effect of two T6SS DNase effectors, namely, Tde1 and Tde4. Both of them function as nuclease effectors, leading to DNA degradation and cell filamentation in prey cells, ultimately resulting in cell death. Our findings deepen our understanding of the T6SS effector repertoires used in P. syringae and will facilitate the development of effective antibacterial strategies.
... The membrane complex built by TssJ, TssL and TssM assembles crossing the inner membrane and defines the position for the T6SS assembly (Durand et al., 2015). The baseplate, formed by the multi-protein complex composed of TssE, TssF, TssG and TssK subunits, docks on the membrane complex and promotes assembly of the sheath-tube complex (Brunet et al., 2015). The tube is assembled by HcPs, forming hexametric rings with a triplet VgrG spike, sharpened by a PAAR protein and enveloped by the sheath complex TssB, TssC (Shneider et al., 2013;Wang et al., 2017). ...
The Type VI Secretion System (T6SS) is used as weapon by a variety of Gram-negative bacteria in polymicrobial niche competition. Its characterization and study gained more interest in recent years. The system functions as a molecular nano-weapon: it is used in inter-kingdom competition by various bacteria to deliver toxic effectors in target cells. In this context, Photorhabdus luminescens subsp. luminescens strain DJC is a microorganism able to colonize different polymicrobial environments, like nematode guts, plant roots and larvae hemolymph. However, the mechanisms used by this microorganism to compete against other bacteria in the same environment have not been clearly described yet. We hypothesis that the T6SS of Photorhabdus luminescens subsp. luminescens strain DJC can play a role in same-niche environments. In this study we focused our attention on the characterization of the T6SS clusters in P. luminescens and its role in this bacteria lifestyle thought bioinformatic, proteomics analyses and inter-bacterial killing assays. Using bioinformatics analysis, we identified four T6SS gene clusters (T6SS-1, T6SS-2, T6SS-3 and T6SS-4) and multiple orphan T6SS related genes in the genome of P. luminescens. Furthermore, we highlighted 11 T6SS effector-immunity pairs, including three undescribed membrane disrupting effectors, each with putatively different antibacterial activities. By comparing the proteomes of P. luminescens wild type cells and the respective isogenic T6SS-deficient strains, we could point out a putative link between T6SS and other P. luminescens related defense mechanisms such as PVCs, T3SS and pyocins. Furthermore, T6SS-deficiency led to a change in phenotypic traits such as motility and secondary metabolism. Our findings shed light on the T6SS of P. luminescens DJC, suggesting a role of the system in the complex life cycle with a putative cross-link with various defense mechanisms and secondary metabolism. This study could help to gain more knowledge on bacterial T6SSs and better understand the P. luminescens ability to live in polymicrobial environments.
... BACTH vectors producing TssF, TssG and GldL C fused to the Bordetella adenylate cyclase T18 or T25 domains have been previously published 6,43 . BACTH plasmid producing PorX Fj and PorX Fj domains fused to the T18 and T25 domains were constructed by restriction free (RF) cloning 53 www.nature.com/scientificreports/ ...
... Bacterial two-hybrid assays were conducted as previously described 43 . The proteins or domains to be tested were fused to the isolated T18 and T25 catalytic domains of the Bordetella adenylate cyclase. ...
... After overnight growth at 28 °C, 15 μL of each culture were spotted onto LB plates supplemented with ampicillin, kanamycin, IPTG, and X-Gal and incubated at 28 °C. Controls include interaction assays with TssF and TssG, two T6SS protein partners from enteroaggregative E. coli 43 unrelated to the T9SS. The experiments were done at least in triplicate and a representative result is shown. ...
The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.
... The T6SS is a harpoon-like contractile nanomachine that triggers interbacterial competition through the injection of toxic effectors directly into target cells in a contact-dependent manner (12,13). This nano-harpoon is composed of a membrane complex and a baseplate that serves as a docking station for the assembly of a tail tube/ sheath complex (14)(15)(16). The tail tube is composed of stacked Hcp hexamers wrapped by a sheath formed by the TssB and TssC proteins (17,18). ...
Bacterial competition may rely on secretion systems such as the type 6 secretion system (T6SS), which punctures and releases toxic molecules into neighboring cells. To subsist, bacterial targets must counteract the threats posed by T6SS-positive competitors. In this study, we used a comprehensive genome-wide high-throughput screening approach to investigate the dynamics of interbacterial competition. Our primary goal was to identify deletion mutants within the well-characterized E. coli K-12 single-gene deletion library, the Keio collection, that demonstrated resistance to T6SS-mediated killing by the enteropathogenic bacterium Cronobacter malonaticus . We identified 49 potential mutants conferring resistance to T6SS and focused our interest on a deletion mutant (∆ fimE ) exhibiting enhanced expression of type 1 fimbriae. We demonstrated that the presence of type 1 fimbriae leads to the formation of microcolonies and thus protects against T6SS-mediated assaults. Collectively, our study demonstrated that adhesive structures such as type 1 fimbriae confer collective protective behavior against T6SS attacks.
IMPORTANCE
Type 6 secretion systems (T6SS) are molecular weapons employed by gram-negative bacteria to eliminate neighboring microbes. T6SS plays a pivotal role as a virulence factor, enabling pathogenic gram-negative bacteria to compete with the established communities to colonize hosts and induce infections. Gaining a deeper understanding of bacterial interactions will allow the development of strategies to control the action of systems such as the T6SS that can manipulate bacterial communities. In this context, we demonstrate that bacteria targeted by T6SS attacks from the enteric pathogen Cronobacter malonaticus , which poses a significant threat to infants, can develop a collective protective mechanism centered on the production of type I fimbriae. These adhesive structures promote the aggregation of bacterial preys and the formation of microcolonies, which protect the cells from T6SS attacks.
... Through its cytoplasmic domain, TssN possesses the only available interface to interact with the BP. TssK is the central component of the BP and in the T6SS i it interacts with the canonical TssJLM MC 24,25 . We thus performed co-production of TssN S and B. fragilis TssK (BF9343_1924) in E. coli and tested their interaction by co-purification (Fig. 4e). ...
... We next sought to determine if TssNQOPR contributed to the assembly of other T6SS iii subcomplexes. One such subcomplex is the sheath, which has been monitored as a readout of comprehensive T6SS assembly since proper sheath polymerization requires both membrane complex and baseplate formation 25 . We constructed superfolder-GFP (sfGFP) fused TssB overexpression strains in a B. fragilis tssB deletion background 27 . ...
... The T6SS baseplate, composed of TssKFGE proteins, makes multiple interactions with the T6SS i MC 25 . Notably, TssK interacts with the TssM and TssL cytoplasmic domains. ...
The type VI secretion system (T6SS) of Gram-negative bacteria inhibits competitor cells through contact-dependent translocation of toxic effector proteins. In Proteobacteria, the T6SS is anchored to the cell envelope through a megadalton-sized membrane complex (MC). However, the genomes of Bacteroidota with T6SSs appear to lack genes encoding homologs of canonical MC components. Here, we identify five genes in Bacteroides fragilis (tssNQOPR) that are essential for T6SS function and encode a Bacteroidota-specific MC. We purify this complex, reveal its dimensions using electron microscopy, and identify a protein-protein interaction network underlying the assembly of the MC including the stoichiometry of the five TssNQOPR components. Protein TssN mediates the connection between the Bacteroidota MC and the conserved baseplate. Although MC gene content and organization varies across the phylum Bacteroidota, no MC homologs are detected outside of T6SS loci, suggesting ancient co-option and functional convergence with the non-homologous MC of Pseudomonadota.
... The type VI secretion system (T6SS) is an important proteinaceous macromolecular apparatus used by many Gram-negative pathogens to deliver toxic proteins (effectors) into target cells (25)(26)(27). A membrane complex (which includes the essential TssM protein) and a baseplate structure assemble within the membrane of cells that possess an active T6SS (28,29). The inner tube of stacked Hcp (hemolysin-coregulated protein) hexamers is surrounded by a contractile outer sheath formed by TssB and TssC proteins (30)(31)(32). ...
Antagonistic behaviors between bacterial cells can have profound effects on microbial populations and disease outcomes. Polymicrobial interactions may be mediated by contact-dependent proteins with antibacterial properties. The type VI secretion system (T6SS) is a macromolecular weapon used by Gram-negative bacteria to translocate proteins into adjacent cells. Many pathogens deploy the T6SS to escape immune cells, eliminate commensal bacteria, and facilitate infection. Stenotrophomonas maltophilia complex is a Gram-negative opportunistic pathogen that primarily affects immunocompromised people and infects the lungs of patients with cystic fibrosis. Infections with the bacterium can be deadly and are challenging to treat because many isolates are multidrug-resistant. We found that globally dispersed S. maltophilia complex clinical and environmental strains possess T6SS genes. We demonstrate that the T6SS of an S. maltophilia complex patient isolate is active and can eliminate other bacteria. We provide evidence that the T6SS contributes to the competitive fitness of S. maltophilia complex against a co-infecting Pseudomonas aeruginosa isolate, and the T6SS alters the organization of S. maltophilia and P. aeruginosa co-cultures. This study expands our knowledge of the mechanisms employed by S. maltophilia complex to secrete antibacterial proteins and compete against other bacteria.
IMPORTANCE
Infections with the opportunistic pathogen Stenotrophomonas maltophilia complex can be fatal for immunocompromised patients. The mechanisms used by the bacterium to compete against other prokaryotes are not well understood. We found that the type VI secretion system (T6SS) allows S. maltophilia complex to eliminate other bacteria and contributes to the competitive fitness against a co-infecting isolate. The presence of T6SS genes in isolates across the globe highlights the importance of this apparatus as a weapon in the antibacterial arsenal of S. maltophilia complex. The T6SS may confer survival advantages to S. maltophilia complex isolates in polymicrobial communities in both environmental settings and during infections.
... The membrane complex recruits the baseplate complex that is required for the coordinated assembly of the tube and sheath components (8,11,13,16). To determine at which step of the T6SS biogenesis PAAR protein is required, we followed the localization of the membrane complex component TssM and the baseplate component TssK by fluorescence microscopy. ...
... We observed the wild-type and PAAR mutant (ΔPAARΩKan) cells producing sfGFP-TssM or TssK-sfGFP (Fig. 5). As described previously, in T6SS-active strains, the sfGFP-TssM and TssK-sfGFP present foci at the membrane [ (8,11), Fig. 5]. We noted that in the absence of PAAR, TssM was correctly localized at the membrane, suggesting that PAAR is not required for proper localization of sfGFP-TssM and thus for membrane complex formation (Fig. 5AB). ...
Bacteria are constantly competing to colonize crowded ecological niches, such as the human gut. The type VI secretion system (T6SS) is a critical bacterial weapon in this warfare. It resembles a crossbow with a poisoned arrow allowing bacteria to inject toxic effectors directly into target cells. This machinery is formed by an envelope-spanning complex which recruits the baseplate, an assembly platform allowing the polymerization of a contractile structure. The tail consists of a tube surrounded by a sheath and topped by the needle complex composed of the VgrG and PAAR proteins. In the enteric pathogen enteroaggregative Escherichia coli (EAEC), the Tle1 phospholipase toxin ensures the antibacterial activity of the T6SS-1. For its transport, Tle1 interacts directly with the trimeric spike protein VgrG. However, the importance and the function of the tip protein PAAR in the T6SS remain unclear. Here, we characterized the PAAR protein of EAEC using biochemical, fluorescence microscopy, and antibacterial competition approaches. Using pull-down assays and cryo-electron microscopy analysis of the (VgrG) 3 -(Tle1) 3 -PAAR complex, we show that PAAR tops and closes the β-prism structure of the VgrG spike. The PAAR protein structure is further tightened by the zinc atom coordinated via conserved residues essential for its function. We provide evidence that PAAR is necessary for T6SS-1-mediated killing due to its requirement for proper T6SS baseplate assembly and further sheath polymerization. Our results suggest that the PAAR protein is an essential component of T6SS.
IMPORTANCE
The type VI secretion system (T6SS) is a bacterial contractile injection system involved in bacterial competition by the delivery of antibacterial toxins. The T6SS consists of an envelope-spanning complex that recruits the baseplate, allowing the polymerization of a contractile tail structure. The tail is a tube wrapped by a sheath and topped by the tip of the system, the VgrG spike/PAAR complex. Effectors loaded onto the puncturing tip or into the tube are propelled in the target cells upon sheath contraction. The PAAR protein tips and sharpens the VgrG spike. However, the importance and the function of this protein remain unclear. Here, we provide evidence for association of PAAR at the tip of the VgrG spike. We also found that the PAAR protein is a T6SS critical component required for baseplate and sheath assembly.
... Most T6SSs include a double membrane spanning complex (TssJLM) which recruits a baseplate complex (TssEFGK) and a spike complex (VgrG heterotrimer, PAAR sharp tip, effectors) (10)(11)(12)(13). Hcp then polymerizes to form the shaft of the projectile, and repeating units of TssBC form a sheath around the shaft that contacts to fire the projectile complex. ...
Soil-borne Ralstonia solanacearum species complex (RSSC) bacteria disrupt rhizosphere and endophytic microbial communities as they invade roots and fatally wilt plants. RSSC pathogens secrete antimicrobial toxins using a type VI secretion system (T6SS). To investigate how evolution and ecology have shaped pathogen T6SS biology, we analyzed the T6SS gene content and architecture across the RSSC pangenome and their evolutionarily relatives. Our analysis reveals that two ecologically similar Burkholderiaceae taxa, xylem pathogenic RSSC bacteria and Acidovorax, have convergently evolved to wield large arsenals of T6SS toxins. To understand the mechanisms underlying genomic enrichment of T6SS toxins, we compiled an atlas of 1,069 auxiliary ("aux") T6SS toxin clusters across 99 high-quality RSSC genomes. We classified 25 types of aux clusters with toxins that predominantly target lipids, nucleic acids, or unknown cellular substrates. The aux clusters were in diverse genetic neighborhoods and had complex phylogenetic distributions, suggesting frequent horizontal gene flow. Phages and other mobile genetic elements account for most of the aux cluster acquisition on the chromosome but very little on the megaplasmid. Nevertheless, RSSC genomes were more enriched in aux clusters on the megaplasmid. Secondary replicons like megaplasmids often evolve more rapidly than the more evolutionarily stable chromosome. Although the single ancestral T6SS was broadly conserved in the RSSC, the T6SS was convergently lost in atypical lineages with vectored transmission. Overall, our data suggest dynamic interplay between the lifestyle of soil-transmitted RSSC lineages and the evolution of T6SSs with robust arsenals of toxins. This pangenomic atlas poises the RSSC as an emerging, tractable model to understand the role of the T6SS in shaping pathogen populations.
... The baseplate (BP) is composed of the highly conserved protein complex TssEFGK and the VgrG-PAAR subunits that will form the spike of the system and upon which polymerization of the tail occurs [43]. The BP also serves as a station for attaching and arranging the spike-attached effectors prior to delivery (Fig. 1ii). ...
... TssE connects to the BP at a later stage and has been suggested to initiate the polymerization of the contractile sheath [22,45]. Direct interactions between the BP components TssE and TssG and the cytosolic domain of TssL and TssM have also been identified [43,50]. These additional contacts, which are not present during BP biogenesis, could be the result of structural rearrangements that stabilize the interactions between the MC and the BP after sheath contraction (Fig. 1v) [22]. ...
... Once anchored, the BP complex acts as the assembly platform for the tail tube/sheath subcomplex with BP components interacting with the inner tube (Hcp hexamers) and the contractile sheath (TssBC) (Fig. 1iii). The model by [22] suggested that TssF interacts with the N-terminal domains of TssBC and that TssFG interacts with Hcp [43]. At the same time, TssG and TssE could stabilize the BP-sheath interface by interacting with the C-terminal domain of TssB [51]. ...
The life of bacteria is challenging, to endure bacteria employ a range of mechanisms to optimize their environment, including deploying the type VI secretion system (T6SS). Acting as a bacterial crossbow, this system delivers effectors responsible for subverting host cells, killing competitors and facilitating general secretion to access common goods. Due to its importance, this lethal machine has been evolutionarily maintained, disseminated and specialized to fulfil these vital functions. In fact, T6SS structural clusters are present in over 25 % of Gram-negative bacteria, varying in number from one to six different genetic clusters per organism. Since its discovery in 2006, research on the T6SS has rapidly progressed, yielding remarkable breakthroughs. The identification and characterization of novel components of the T6SS, combined with biochemical and structural studies, have revealed fascinating mechanisms governing its assembly, loading, firing and disassembly processes. Recent findings have also demonstrated the efficacy of this system against fungal and Gram-positive cells, expanding its scope. Ongoing research continues to uncover an extensive and expanding repertoire of T6SS effectors, the genuine mediators of T6SS function. These studies are shedding light on new aspects of the biology of prokaryotic and eukaryotic organisms. This review provides a comprehensive overview of the T6SS, highlighting recent discoveries of its structure and the diversity of its effectors. Additionally, it injects a personal perspective on avenues for future research, aiming to deepen our understanding of this combative system.
... In this study, the ΔFur mutant strain showed suppressed bio lm formation, suggesting that Fur is a negative regulator of bio lm formation in V. harveyi and it possible play an indirect role by regulating the expression levels of type II secretion systems (T2SS) and type VI secretion systems (T6SS)(Smith et al., 2021). Furthermore, the RNA-seq showed the expression of some genes (e.g., tssM, LA59_RS08435, gspL) which regulated the T2SS and T6SS(Brunet et al., 2015; Michel-Souzy et al., 2018) were signi cantly decreased(Fig. 8). ...
Vibrio harveyi is commonly found in salt and brackish water, and is recognized as a serious bacterial pathogen in aquaculture worldwide. In this study, we cloned the ferric uptake regulator ( Fur ) gene from V. harveyi wild-type strain HA_1, which was isolated from diseased American eels ( Anguilla rostrata ) and has a length of 450 bp, encoding 149 amino acids. Then, a mutant strain, HA_1-Δ Fur , was constructed through homologous recombination of a suicide plasmid (pCVD442). The HA_1-Δ Fur mutant exhibited attenuated biofilm formation, intensified swarming motility, and 18-fold decrease (5.5%) in virulence to the American eels, but it showing no difference in growth and hemolysis with the wild-type strain. Transcriptome analysis revealed that 875 genes were differentially expressed in the Δ Fur mutant, with 385 up-regulated and 490 down-regulated DEGs. GO and KEGG enrichment analysis revealed that, compared to the wild-type strain, the type II secretion systems (T2SS), type VI secretion systems (T6SS), amino acid synthesis and transport, and energy metabolism pathways were significantly down-regulated, but the ABC transporters and biosynthesis of siderophore group non-ribosomal peptides pathways were up-regulated in the Δ Fur strain. The qRT-PCR results further confirmed that DEGs responsible for amino acid transport and energy metabolism were positively regulated, but DEGs involved in iron acquisition were negatively regulated in the Δ Fur strain. These findings suggest that the gene Fur contribute to the virulence of V. harveyi through biofilm formation, energy metabolism, and transcript regulation.
