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The type VI secretion system (T6SS) is an important mediator of microbe–microbe and microbe–host interactions. Gram-negative bacteria use the T6SS to inject T6SS effectors (T6Es), which are usually proteins with toxic activity, into neighboring cells. Antibacterial effectors have cognate immunity proteins that neutralize self-intoxication. Here, we applied novel structural bioinformatic tools to perform systematic discovery and functional annotation of T6Es and their cognate immunity proteins from a dataset of 17,920 T6SS-encoding bacterial genomes. Using structural clustering, we identified 517 putative T6E families, outperforming sequence-based clustering. We developed a logistic regression model to reliably quantify protein–protein interaction of new T6E-immunity pairs, yielding candidate immunity proteins for 231 out of the 517 T6E families. We used sensitive structure-based annotation which yielded functional annotations for 51% of the T6E families, again outperforming sequence-based annotation. Next, we validated four novel T6E-immunity pairs using basic experiments in E. coli. In particular, we showed that the Pfam domain DUF3289 is a homolog of Colicin M and that DUF943 acts as its cognate immunity protein. Furthermore, we discovered a novel T6E that is a structural homolog of SleB, a lytic transglycosylase, and identified a specific glutamate that acts as its putative catalytic residue. Overall, this study applies novel structural bioinformatic tools to T6E-immunity pair discovery, and provides an extensive database of annotated T6E-immunity pairs. Synopsis: Structural bioinformatic tools were utilized for the discovery of novel specialized Type VI Secretion System (T6SS) effectors and their cognate immunity proteins, highlighting their utility over standard sequence-based tools. The effector predictions were supported by experimental results. Structural clustering provided better compression of effectors than sequence-based methods, with 517 structural clusters representing the structure space of specialized effectors in Proteobacteria. The ipTM score from Alphafold-multimer was used as a reliable and quantitative measure for predicting candidate immunity proteins in 231 out of 517 effector clusters. Annotations were provided for 265 out of the 517 specialized effector domain families using fast and sensitive searches with Foldseek, expanding capabilities beyond Pfam-based annotation alone. Four putative effectors were demonstrated to be toxic to Escherichia coli, with co-expression of cognate immunity proteins neutralizing their toxicity. Structural bioinformatic tools were utilized for the discovery of novel specialized Type VI Secretion System (T6SS) effectors and their cognate immunity proteins, highlighting their utility over standard sequence-based tools. The effector predictions were supported by experimental results. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) of many gram‐negative bacteria injects toxic effectors into adjacent cells to manipulate host cells during pathogenesis or to kill competing bacteria. However, the identification and function of the T6SS effectors remains only partly known. Pantoea ananatis, a gram‐negative bacterium, is commonly found in various plants and natural environments, including water and soil. In the current study, genomic analysis of P. ananatis DZ‐12 causing brown stalk rot on maize demonstrated that it carries three T6SS gene clusters, namely, T6SS‐1, T6SS‐2, and T6SS‐3. Interestingly, only T6SS‐1 secretion systems are involved in pathogenicity and bacterial competition. The study also investigated the T6SS‐1 system in detail and identified an unknown T6SS‐1‐secreted effector TseG by using the upstream T6SS effector chaperone TecG containing a conserved domain of DUF2169. TseG can directly interact with the chaperone TecG for delivery and with a downstream immunity protein TsiG for protection from its toxicity. TseG, highly conserved in the Pantoea genus, is involved in virulence in maize, potato, and onion. Additionally, P. ananatis uses TseG to target Escherichia coli, gaining a competitive advantage. This study provides the first report on the T6SS‐1‐secreted effector from P. ananatis, thereby enriching our understanding of the various types and functions of type VI effector proteins. [ABSTRACT FROM AUTHOR]

The Type VI Secretion System (T6SS) is a widespread and highly effective mechanism of microbial warfare; it confers the ability to efficiently kill susceptible cells within close proximity. Due to its large physical size, complexity, and ballistic basis for intoxication, it has widely been assumed to incur significant growth costs in the absence of improved competitive outcomes. In this study, we precisely examine the fitness costs of constitutive T6SS firing in the bacterium Vibrio cholerae. We find that, contrary to expectations, constitutive expression of the T6SS has a negligible impact on growth, reducing growth fitness by 0.025 ± 0.5% (95% CI) relative to a T6SS− control. Mathematical modeling of microbial populations demonstrates that, due to clonal interference, constitutive expression of the T6SS will often be neutral, with little impact on evolutionary outcomes. Our findings underscore the importance of precisely measuring the fitness costs of microbial social behaviors and help explain the prevalence of the T6SS across Gram‐negative bacteria. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is a powerful bacterial molecular weapon that can inject effector proteins into prokaryotic or eukaryotic cells, thereby participating in the competition between bacteria and improving bacterial environmental adaptability. Although most current studies of the T6SS have focused on animal bacteria, this system is also significant for the adaptation of plant-associated bacteria. This paper briefly introduces the structure and biological functions of the T6SS. We summarize the role of plant-associated bacterial T6SS in adaptability to host plants and the external environment, including resistance to biotic stresses such as host defenses and competition from other bacteria. We review the role of the T6SS in response to abiotic factors such as acid stress, oxidation stress, and osmotic stress. This review provides an important reference for exploring the functions of the T6SS in plant-associated bacteria. In addition, characterizing these anti-stress functions of the T6SS may provide new pathways toward eliminating plant pathogens and controlling agricultural losses. [ABSTRACT FROM AUTHOR]

Edwardsiella ictaluri is a Gram-negative facultative intracellular fish pathogen causing enteric septicemia of catfish (ESC). While various secretion systems contribute to E. ictaluri virulence, the Type VI secretion system (T6SS) remains poorly understood. In this study, we constructed 13 E. ictaluri T6SS mutants using splicing by overlap extension PCR and characterized them, assessing their uptake and survival in channel catfish (Ictalurus punctatus) peritoneal macrophages, attachment and invasion in channel catfish ovary (CCO) cells, in vitro stress resistance, and virulence and efficacy in channel catfish. Among the mutants, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO exhibited reduced replication inside peritoneal macrophages. EiΔevpM, EiΔevpN, and EiΔevpO showed significantly decreased attachment to CCO cells, while EiΔevpN and EiΔevpO also displayed reduced invasion of CCO cells (p < 0.05). Overall, T6SS mutants demonstrated enhanced resistance to oxidative and nitrosative stress in the nutrient-rich medium compared to the minimal medium. However, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO were susceptible to oxidative stress in both nutrient-rich and minimal medium. In fish challenges, EiΔevpD, EiΔevpE, EiΔevpG, EiΔevpJ, and EiΔevpK exhibited attenuation and provided effective protection against E. ictaluri wild-type (EiWT) infection in catfish fingerlings. However, their attenuation and protective efficacy were lower in catfish fry. These findings shed light on the role of the T6SS in E. ictaluri pathogenesis, highlighting its significance in intracellular survival, host cell attachment and invasion, stress resistance, and virulence. The attenuated T6SS mutants hold promise as potential candidates for protective immunization strategies in catfish fingerlings. [ABSTRACT FROM AUTHOR]

Bacteria communicate with their surroundings through diverse secretory systems, and the recently discovered Type VI Secretion System (T6SS) has gained significant attention. Klebsiella pneumoniae (K. pneumoniae), an opportunistic pathogen known for causing severe infections in both hospital and animal settings, possesses this intriguing T6SS. This system equips K. pneumoniae with a formidable armory of protein-based weaponry, enabling the delivery of toxins into neighboring cells, thus granting a substantial competitive advantage. Remarkably, the T6SS has also been associated with K. pneumoniae's ability to form biofilms and acquire resistance against antibiotics. However, the precise effects of the T6SS on K. pneumoniae's functions remain inadequately studied, despite research efforts to understand the intricacies of these mechanisms. This comprehensive review aims to provide an overview of the current knowledge regarding the biological functions and regulatory mechanisms of the T6SS in K. pneumoniae. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is used by bacteria for virulence, resistance to grazing, and competition with other bacteria. We previously demonstrated that the role of the T6SS in interbacterial competition and in resistance to grazing is enhanced in Vibrio cholerae in the presence of subinhibitory concentrations of polymyxin B. Here, we performed a global quantitative proteomic analysis and a targeted transcriptomic analysis of the T6SS-known regulators in V. cholerae grown with and without polymyxin B. The proteome of V. cholerae is greatly modified by polymyxin B with more than 39% of the identified cellular proteins displaying a difference in their abundance, including T6SS-related proteins. We identified a regulator whose abundance and expression are increased in the presence of polymyxin B, vxrB, the response regulator of the two-component system VxrAB (VCA0565-66). In vxrAB, vxrA and vxrB deficient mutants, the expression of both hcp copies (VC1415 and VCA0017), although globally reduced, was not modified by polymyxin B. These hcp genes encode an identical protein Hcp, which is the major component of the T6SS syringe. Thus, the upregulation of the T6SS in the presence of polymyxin B appears to be, at least in part, due to the two-component system VxrAB. [ABSTRACT FROM AUTHOR]

A. baumannii can rapidly acquire new resistance mechanisms and persist on abiotic surface, enabling the colonization of asymptomatic human host. In Acinetobacter the type VI secretion system (T6SS) is involved in twitching, surface motility and is used for interbacterial competition allowing the bacteria to uptake DNA. A. baumannii possesses a T6SS that has been well studied for its regulation and specific activity, but little is known concerning its assembly and architecture. The T6SS nanomachine is built from three architectural sub-complexes. Unlike the baseplate (BP) and the tail-tube complex (TTC), which are inherited from bacteriophages, the membrane complex (MC) originates from bacteria. The MC is the most external part of the T6SS and, as such, is subjected to evolution and adaptation. One unanswered question on the MC is how such a gigantesque molecular edifice is inserted and crosses the bacterial cell envelope. The A. baumannii MC lacks an essential component, the TssJ lipoprotein, which anchors the MC to the outer membrane. In this work, we studied how A. baumannii compensates the absence of a TssJ. We have characterized for the first time the A. baumannii's specific T6SS MC, its unique characteristic, its membrane localization, and assembly dynamics. We also defined its composition, demonstrating that its biogenesis employs three Acinetobacter-specific envelope-associated proteins that define an intricate network leading to the assembly of a five-proteins membrane super-complex. Our data suggest that A. baumannii has divided the function of TssJ by (1) co-opting a new protein TsmK that stabilizes the MC and by (2) evolving a new domain in TssM for homo-oligomerization, a prerequisite to build the T6SS channel. We believe that the atypical species-specific features we report in this study will have profound implication in our understanding of the assembly and evolutionary diversity of different T6SSs, that warrants future investigation. Author summary: Assembly of envelope-spanning secretion nanomachines requires the concerted action of architectural proteins whose conservation have helped their identification in bacterial genomes. Specific adaptation occurs to fine tune the nanomachine to specific bacterial environment or biology. The type VI secretion system (T6SS) assembles a trans-envelope channel, the membrane complex (MC) that plays a key role in the transport of the toxin-loaded speargun. Even though the evolutionary pressure has maintained a high degree of conservation of this complex across species, here we demonstrated that Acinetobacter baumannii has evolved two new players to compensate the absence of the highly conserved TssJ lipoprotein. Our finding improves our understanding of the key roles of the lipoprotein: MC stability and multimerization. Understanding the molecular evolution of nanomachines offers the possibility to better understand their architecture and functioning, but also for pathogenic bacteria to identify specific drug target to block their virulence. [ABSTRACT FROM AUTHOR]

Pseudomonas syringae pv. aptata is a member of the sugar beet pathobiome and the causative agent of leaf spot disease. Like many pathogenic bacteria, P. syringae relies on the secretion of toxins, which manipulate host-pathogen interactions, to establish and maintain an infection. This study analyzes the secretome of six pathogenic P. syringae pv. aptata strains with different defined virulence capacities in order to identify common and strain-specific features, and correlate the secretome with disease outcome. All strains show a high type III secretion system (T3SS) and type VI secretion system (T6SS) activity under apoplast-like conditions mimicking the infection. Surprisingly, we found that low pathogenic strains show a higher secretion of most T3SS substrates, whereas a distinct subgroup of four effectors was exclusively secreted in medium and high pathogenic strains. Similarly, we detected two T6SS secretion patterns: while one set of proteins was highly secreted in all strains, another subset consisting of known T6SS substrates and previously uncharacterized proteins was exclusively secreted in medium and high virulence strains. Taken together, our data show that P. syringae pathogenicity is correlated with the repertoire and fine-tuning of effector secretion and indicate distinct strategies for establishing virulence of P. syringae pv. aptata in plants. [ABSTRACT FROM AUTHOR]

Bacterial secretion systems are nanomolecular complexes that release a diverse set of virulence factors/or proteins into its surrounding or translocate to their target host cells. Among these systems, type VI secretion system 'T6SS' is a recently discovered molecular secretion system which is widely distributed in Gram-negative (−ve) bacteria, and shares structural similarity with the puncturing device of bacteriophages. The presence of T6SS is an advantage to many bacteria as it delivers toxins to its neighbour pathogens for competitive survival, and also translocates protein effectors to the host cells, leading to disruption of lipid membranes, cell walls, and cytoskeletons etc. Recent studies have characterized both anti-prokaryotic and anti-eukaryotic effectors, where T6SS is involved in diverse cellular functions including favouring colonization, enhancing the survival, adhesive modifications, internalization, and evasion of the immune system. With the evolution of advanced genomics and proteomics tools, there has been an increase in the number of characterized T6SS effector arsenals and also more clear information about the adaptive significance of this complex system. The functions of T6SS are generally regulated at the transcription, post-transcription and post-translational levels through diverse mechanisms. In the present review, we aimed to provide information about the distribution of T6SS in diverse bacteria, any structural similarity/or dissimilarity, effectors proteins, functional significance, and regulatory mechanisms. We also tried to provide information about the diverse roles played by T6SS in its natural environments and hosts, and further any changes in the microbiome. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is a secretion apparatus widely found in pathogenic Gram-negative bacteria and is important for competition among various bacteria and host cell pathogenesis. Hcp is a core component of functional T6SS and transports toxic effectors into target cells by assembling to form tube-like structures. Studies have shown that Hcp simultaneously acts as an effector to influence cellular physiological activities; however, the mechanism of its activity in host cells remains unclear. To investigate the target of effector protein Hcp2a in a chicken fibroblast cell line, we first detected the subcellular localization of Hcp2a in DF-1 cells by indirect immunofluorescence assay. The results showed that Hcp2a protein was localized in the endoplasmic reticulum of DF-1 cells. We also used a streptavidin–biotin affinity pull-down assay combined with LC–MS/MS to screen DF-1 cell lysates for proteins that interact with Hcp2a and analyze the cellular functional pathways affected by them. The results showed that Hcp2a interacted with 52 DF-1 cellular proteins that are involved in multiple intracellular pathways. To further explore the mechanism of Hcp2a protein targeting the endoplasmic reticulum of DF-1 cells, we screened three endoplasmic reticulum-associated proteins (RSL1D1, RPS3A, and RPL23) from 52 prey proteins of Hcp2a for protein–protein molecular docking analysis. The docking analysis showed that the effector protein Hcp2a and the RPL23 protein had good complementarity. Overall, we propose that Hcp2a has strong binding activity to the RPL23 protein in DF-1 cells and this may help Hcp2a anchor to the endoplasmic reticulum in DF-1 cells. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is likely the most common bacterial cause of gastroenteritis worldwide, responsible for millions of cases of inflammatory diarrhea characterized by severe abdominal cramps and blood in the stool. Further, C. jejuni infections are associated with post-infection sequelae in developed countries and malnutrition and growth-stunting in low- and middle-income countries. Despite the increasing prevalence of the disease, campylobacteriosis, and the recognition that this pathogen is a serious health threat, our understanding of C. jejuni pathogenesis remains incomplete. In this review, we focus on the Campylobacter secretion systems proposed to contribute to host-cell interactions and survival in the host. Moreover, we have applied a genomics approach to defining the structural and mechanistic features of C. jejuni type III, IV, and VI secretion systems. Special attention is focused on the flagellar type III secretion system and the prediction of putative effectors, given that the proteins exported via this system are essential for host cell invasion and the inflammatory response. We conclude that C. jejuni does not possess a type IV secretion system and relies on the type III and type VI secretion systems to establish a niche and potentiate disease. [ABSTRACT FROM AUTHOR]

Many bacteria of the genus Bradyrhizobium are capable of inducing nodules in legumes. In this work, the importance of a type VI secretion system (T6SS) in a symbiotic strain of the genus Bradyrhizobium is described. T6SS of Bradyrhizobium sp. LmicA16 (A16) is necessary for efficient nodulation with Lupinus micranthus and Lupinus angustifolius. A mutant in the gene vgrG, coding for a component of the T6SS nanostructure, induced less nodules and smaller plants than the wild-type (wt) strain and was less competitive when co-inoculated with the wt strain. A16 T6SS genes are organized in a 26-kb DNA region in two divergent gene clusters of nine genes each. One of these genes codes for a protein (Tsb1) of unknown function but containing a methyltransferase domain. A tsb1 mutant showed an intermediate symbiotic phenotype regarding vgrG mutant and higher mucoidity than the wt strain in free-living conditions. T6SS promoter fusions to the lacZ reporter indicate expression in nodules but not in free-living cells grown in different media and conditions. The analysis of nodule structure revealed that the level of nodule colonization was significantly reduced in the mutants with respect to the wt strain. [ABSTRACT FROM AUTHOR]

A type VI secretion system (T6SS) gene cluster has been reported in Acidovorax citrulli. Research on the activation conditions, functions, and the interactions between key elements in A. citrulli T6SS is lacking. Hcp (Hemolysin co-regulated protein) is both a structural protein and a secretion protein of T6SS, which makes it a special element. The aims of this study were to determine the role of Hcp and its activated conditions to reveal the functions of T6SS. In virulence and colonization assays of hcp deletion mutant strain Δhcp, tssm (type VI secretion system membrane subunit) deletion mutant strain Δtssm and double mutant ΔhcpΔtssm, population growth was affected but not virulence after injection of cotyledons and seed-to-seedling transmission on watermelon. The population growth of Δhcp and Δtssm were lower than A. citrulli wild type strain Aac5 of A. citrulli group II at early stage but higher at a later stage. Deletion of hcp also affected growth ability in different culture media, and the decline stage of Δhcp was delayed in KB medium. Biofilm formation ability of Δhcp, Δtssm and ΔhcpΔtssm was lower than Aac5 with competition by prey bacteria but higher in KB and M9-Fe3+ medium. Deletion of hcp reduced the competition and survival ability of Aac5. Based on the results of Western blotting and qRT-PCR analyses, Hcp is activated by cell density, competition, ferric irons, and the host plant. The expression levels of genes related to bacterial secretion systems, protein export, and several other pathways, were significantly changed in the Δhcp mutant compared to Aac5 when T6SS was activated at high cell density. Based on transcriptome data, we found that a few candidate effectors need further identification. The phenotypes, activated conditions and transcriptome data all supported the conclusion that although there is only one T6SS gene cluster present in the A. citrulli group II strain Aac5, it related to multiple biological processes, including colonization, growth ability, competition and biofilm formation. [ABSTRACT FROM AUTHOR]

Equipped with a plethora of secreted toxic effectors, protein secretion systems are essential for bacteria to interact with and manipulate their neighboring environment to survive in host microbiota and other highly competitive communities. While effectors have received spotlight attention in secretion system studies, many require accessory chaperone and adaptor proteins for proper folding/unfolding and stability throughout the secretion process. Here, we review the functions of chaperones and adaptors of three protein secretions systems, type 3 secretion system (T3SS), type 4 secretion system (T4SS), and type 6 secretion system (T6SS), which are employed by many Gram‐negative bacterial pathogens to deliver toxins to bacterial, plant, and mammalian host cells through direct contact. Since chaperone and adaptor functions of the T3SS and the T4SS are relatively well studied, we discuss in detail the methods of chaperone‐facilitated effector secretion by the T6SS and highlight commonalities between the effector chaperone/adaptor proteins of these diverse secretion systems. While the chaperones and adaptors are generally referred to as accessory proteins as they are not directly involved in toxicities to target cells, they are nonetheless vital for the biological functions of the secretion systems. Future research on biochemical and structural properties of these chaperones will not only elucidate the mechanisms of chaperone‐effector binding and release process but also facilitate custom design of cargo effectors to be translocated by these widespread secretion systems for biotechnological applications. [ABSTRACT FROM AUTHOR]

Bacteria require a number of systems, including the type VI secretion system (T6SS), for interbacterial competition and pathogenesis. The T6SS is a large nanomachine that can deliver toxins directly across membranes of proximal target cells. Since major reassembly of T6SS is necessary after each secretion event, accurate timing and localization of T6SS assembly can lower the cost of protein translocation. Although critically important, mechanisms underlying spatiotemporal regulation of T6SS assembly remain poorly understood. Here, we used super‐resolution live‐cell imaging to show that while Acinetobacter and Burkholderia thailandensis can assemble T6SS at any site, a significant subset of T6SS assemblies localizes precisely to the site of contact between neighboring bacteria. We identified a class of diverse, previously uncharacterized, periplasmic proteins required for this dynamic localization of T6SS to cell–cell contact (TslA). This precise localization is also dependent on the outer membrane porin OmpA. Our analysis links transmembrane communication to accurate timing and localization of T6SS assembly as well as uncovers a pathway allowing bacterial cells to respond to cell–cell contact during interbacterial competition. Synopsis: The assembly of the Type VI secretion system in Acinetobacter and Burkholderia can be precisely positioned to the site of a cell–cell contact allowing for efficient toxin translocation during interbacterial competition. Super‐resolution microscopy shows that a significant subset of T6SS assemblies initiate at the site of a contact between neighboring bacteria.T6SS baseplate and membrane complex is required for the formation of small sheath foci followed by delayed sheath polymerization.A previously uncharacterized periplasmic protein TslA and the outer membrane porin OmpA are required for localization of T6SS assembly at the cell–cell contact sites. [ABSTRACT FROM AUTHOR]

Sheath proteins comprise a part of the contractile molecular machinery present in bacteriophages with myoviral morphology, contractile injection systems, and the type VI secretion system (T6SS) found in many Gram-negative bacteria. Previous research on sheath proteins has demonstrated that they share common structural features, even though they vary in their size and primary sequence. In this study, 112 contractile phage tail sheath proteins (TShP) representing different groups of bacteriophages and archaeal viruses with myoviral morphology have been modelled with the novel machine learning software, AlphaFold 2. The obtained structures have been analysed and conserved and variable protein parts and domains have been identified. The common core domain of all studied sheath proteins, including viral and T6SS proteins, comprised both N-terminal and C-terminal parts, whereas the other parts consisted of one or several moderately conserved domains, presumably added during phage evolution. The conserved core appears to be responsible for interaction with the tail tube protein and assembly of the phage tail. Additional domains may have evolved to maintain the stability of the virion or for adsorption to the host cell. Evolutionary relations between TShPs representing distinct viral groups have been proposed using a phylogenetic analysis based on overall structural similarity and other analyses. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is a nosocomial opportunistic pathogen that can cause pneumonia, liver abscesses, and infections of the bloodstream. The resistance and pathogenicity of K. pneumoniae pose major challenges to clinical practice. However, the ecology and pathogenic mechanisms of K. pneumoniae have not been fully elucidated. Among these mechanisms, the secretion systems encoded by strains of the bacteria confer adaptive advantages depending on the niche occupied. The type VI secretion system (T6SS) is a multi-protein complex that delivers effector proteins to the extracellular environment or directly to eukaryotic or prokaryotic cells. T6SSs are widely distributed in Gram-negative bacteria and play an important role in bacterial virulence and the interactions between bacteria and other microorganisms or the environment. This study aimed to enhance the understanding of the characteristics of T6SSs in K. pneumoniae through an in-depth comparative genomic analysis of the T6SS in 241 sequenced strains of K. pneumoniae. We identified the T6SS loci, the synteny of the loci in different species, as well as the effectors and core T6SS-related genes in K. pneumoniae. The presence of a T6SS was a common occurrence in K. pneumoniae , and two T6SS clusters are the most prevalent. The variable region downstream of the gene vgrG usually encodes effector proteins. Conserved domain analysis indicated that the identified putative effectors in K. pneumoniae had the functions of lipase, ribonuclease, deoxyribonuclease, and polysaccharide hydrolase. However, some effectors did not contain predicted functional domains, and their specific functions have yet to be elucidated. This in silico study represents a detailed analysis of T6SS-associated genes in K. pneumoniae and provides a foundation for future studies on the mechanism(s) of T6SSs, especially effectors, which may generate new insights into pathogenicity and lead to the identification of proteins with novel antimicrobial properties. [ABSTRACT FROM AUTHOR]

Marine bacteria can cause harm to single-celled and multicellular eukaryotes. However, relatively little is known about the underlying genetic basis for marine bacterial interactions with higher organisms. We examined whole-genome sequences from a large number of marine bacteria for the prevalence of homologues to virulence genes and pathogenicity islands known from bacteria that are pathogenic to terrestrial animals and plants. As many as 60 out of 119 genomes of marine bacteria, with no known association to infectious disease, harboured genes of virulence-associated types III, IV, V and VI protein secretion systems. Type III secretion was relatively uncommon, while type IV was widespread among alphaproteobacteria (particularly among roseobacters) and type VI was primarily found among gammaproteobacteria. Other examples included homologues of the Yersinia murine toxin and a phage-related ‘antifeeding’ island. Analysis of the Global Ocean Sampling metagenomic data indicated that virulence genes were present in up to 8% of the planktonic bacteria, with highest values in productive waters. From a marine ecology perspective, expression of these widely distributed genes would indicate that some bacteria infect or even consume live cells, that is, generate a previously unrecognized flow of organic matter and nutrients directly from eukaryotes to bacteria. [ABSTRACT FROM AUTHOR]

Rhizosphere colonizing plant growth promoting bacteria (PGPB) increase their competitiveness by producing diffusible toxic secondary metabolites, which inhibit competitors and deter predators. Many PGPB also have one or more Type VI Secretion System (T6SS), for the delivery of weapons directly into prokaryotic and eukaryotic cells. Studied predominantly in human and plant pathogens as a virulence mechanism for the delivery of effector proteins, the function of T6SS for PGPB in the rhizosphere niche is poorly understood. We utilized a collection of Pseudomonas chlororaphis 30–84 mutants deficient in one or both of its two T6SS and/or secondary metabolite production to examine the relative importance of each T6SS in rhizosphere competence, bacterial competition, and protection from bacterivores. A mutant deficient in both T6SS was less persistent than wild type in the rhizosphere. Both T6SS contributed to competitiveness against other PGPB or plant pathogenic strains not affected by secondary metabolite production, but only T6SS-2 was effective against strains lacking their own T6SS. Having at least one T6SS was also essential for protection from predation by several eukaryotic bacterivores. In contrast to diffusible weapons that may not be produced at low cell density, T6SS afford rhizobacteria an additional, more immediate line of defense against competitors and predators. [ABSTRACT FROM AUTHOR]

Background: Klebsiella pneumoniae (K. pneumoniae) causes bloodstream infection (BSI), which is responsible for a high rate of morbidity and mortality among different populations. In mainland China, data on the correlation and features of the type VI secretion system (T6SS) gene cluster in K. pneumoniae is currently scarce. As a result, we conducted a prospective investigation to determine the involvement of the T6SS in K. pneumoniae pathogenicity and antibiotic resistance. Methods: In this prospective analysis, we enrolled 119 individuals who had been diagnosed with K. pneumoniae bloodstream infection between July 2019 and January 2021 and acquired demographic and clinical data from their medical records. The virulence genes rmpA, rmpA2, aerobactin, iroB, hcp, vgrG, and icmF were tested for K1 and K2, antimicrobial susceptibility. Five T6SS-positive and five T6SS-negative isolates were chosen for the competition, serum resistance, and biofilm formation experiments to further gain insights regarding the microbiological properties of T6SS-positive K. pneumoniae isolates. Results: Among 119 isolates obtained from patients with BSIs, 20 (16.8%) were T6SS positive K. pneumoniae. T6SS positive strains had four virulence genes and a greater K1 capsular serotypes rate than T6SS negative bacteria. Among hvKP isolates, the T6SS positive rate was substantially greater than the T6SS negative rate (P = 0.001). T6SS-positive K. pneumoniae strains had a lower rate of antimicrobial resistance in comparison to T6SS-negative bacteria. T6SS-positive isolates may be more competitive with Escherichia coli than T6SS-negative isolates. T6SS-positive isolates, on the other hand, did not show stronger biofilm-forming activity or a higher survival rate in the presence of normal human serum in comparison to T6SS-negative isolates. Conclusion: T6SS-positive K. pneumoniae was common in people who had BSIs. In T6SS‐containing K. pneumoniae, the system may play a major role in bacterial competition. [ABSTRACT FROM AUTHOR]

The Type VI Secretion System (T6SS) is a multiprotein device that has emerged as an important fitness and virulence factor for many Gram-negative bacteria through the injection of effector proteins into prokaryotic or eukaryotic cells via a contractile mechanism. While some effector proteins specifically target bacterial or eukaryotic cells, others can target both types of cells (trans-kingdom effectors). In Salmonella , five T6SS gene clusters have been identified within pathogenicity islands SPI-6, SPI-19, SPI-20, SPI-21, and SPI-22, which are differentially distributed among serotypes. Salmonella enterica serotype Dublin (S. Dublin) is a cattle-adapted pathogen that harbors both T6SSSPI-6 and T6SSSPI-19. Interestingly, while both systems have been linked to virulence and host colonization in S. Dublin, an antibacterial activity has not been detected for T6SSSPI-6 in this serotype. In addition, there is limited information regarding the repertoire of effector proteins encoded within T6SSSPI-6 and T6SSSPI-19 gene clusters in S. Dublin. In the present study, we demonstrate that T6SSSPI-6 and T6SSSPI-19 of S. Dublin CT_02021853 contribute to interbacterial competition. Bioinformatic and comparative genomic analyses allowed us to identify genes encoding three candidate antibacterial effectors located within SPI-6 and two candidate effectors located within SPI-19. Each antibacterial effector gene is located upstream of a gene encoding a hypothetic immunity protein, thus conforming an effector/immunity (E/I) module. Of note, the genes encoding these effectors and immunity proteins are widely distributed in Salmonella genomes, suggesting a relevant role in interbacterial competition and virulence. Finally, we demonstrate that E/I modules SED_RS01930/SED_RS01935 (encoded in SPI-6), SED_RS06235/SED_RS06230, and SED_RS06335/SED_RS06340 (both encoded in SPI-19) contribute to interbacterial competition in S. Dublin CT_02021853. [ABSTRACT FROM AUTHOR]

Bacterial type III secretions system (T3SS) is a membrane embedded needle like macromolecular complex structure present in Gram negative bacteria and mostly found in Yersinia, Salmonella, Shigella, Entero Pathogenic E. coli (EPEC), Entero Haemorrhagic E. coli (EHEC) and Pseudomonas aeruginosa. The infection dynamic involves several sub cellular components for easy delivery of the effector molecules from bacteria to the host cells to survive the host immune mechanism. Mainly three categories of proteins are involved-structural, translocator and effector proteins. T3SSs can be classified into seven phylogenetic families, based on the genetic analysis of their components. Multiple T3SSs are found in the same bacteria with the purpose of causing infection in multiple steps. Their contribution to virulence mechanism is mainly through modification of the host cytoskeleton system or interfering with the signaling Pathways in the host cellular events related to the defensive mechanism. The infection requires multi-step regulatory strategies which include spatiotemporal regulation of a different set of effector proteins encoded genes. Despite their contribution in virulence mechanism, they can be utilized by re-engineering them to deliver either various therapeutic protein agents or could be used as an alternative novel approach for antigen delivery into the host. Apart from its use as the delivery platform they can be targeted using broad spectrum inhibitors against diverse sets of T3SS mediated diseases. Therefore, this review summarizes the basic structure, its regulatory mechanism in different bacteria and the future perspective. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 Å resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise β-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs 'cocoon' where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal 'plug' domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS. Author summary: Bacteria have developed a variety of strategies to compete for nutrients and limited resources. One system widely used by Gram-negative bacteria is the T6 secretion system which delivers a plethora of effectors into competing bacterial cells. Known functions of effectors are degradation of the cell wall, the depletion of essential metabolites such as NAD+ or the cleavage of DNA. RhsA is an effector from the widespread plant-protecting bacteria Pseudomonas protegens. We found that RhsA forms a closed cocoon similar to that found in bacterial Tc toxins and metazoan teneurin proteins. The effector cleaves its polypeptide chain by itself in three pieces, namely the N-terminal domain including a seal, the cocoon and the actual toxic component which potentially cleaves DNA. The toxic component is encapsulated in the large cocoon, so that the effector producing bacterium is protected from the toxin. In order for the toxin to exit the cocoon, we propose that the seal, which closes the cocoon at one end, is removed by mechanical forces during injection of the effector by the T6 secretion system. We further hypothesize about different scenarios for the delivery of the toxin into the cytoplasm of the host cell. Together, our findings expand the knowledge of the mechanism of action of the T6 secretion system and its essential role in interbacterial competition. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is a spear-like nanomachine found in gram-negative pathogens for delivery of toxic effectors to neighboring bacterial and host cells. Its assembly requires a tip spike complex consisting of a VgrG-trimer, a PAAR protein, and the interacting effectors. However, how the spike controls T6SS assembly remains elusive. Here we investigated the role of three VgrG-effector pairs in Aeromonas dhakensis strain SSU, a clinical isolate with a constitutively active T6SS. By swapping VgrG tail sequences, we demonstrate that the C-terminal ~30 amino-acid tail dictates effector specificity. Double deletion of vgrG1&2 genes (VgrG3+) abolished T6SS secretion, which can be rescued by ectopically expressing chimeric VgrG3 with a VgrG1/2-tail but not the wild type VgrG3. In addition, deletion of effector-specific chaperones also severely impaired T6SS secretion, despite the presence of intact VgrG and effector proteins, in both SSU and Vibrio cholerae V52. We further show that SSU could deliver a V. cholerae effector VasX when expressing a plasmid-borne chimeric VgrG with VasX-specific VgrG tail and chaperone sequences. Pull-down analyses show that two SSU effectors, TseP and TseC, could interact with their cognate VgrGs, the baseplate protein TssK, and the key assembly chaperone TssA. Effectors TseL and VasX could interact with TssF, TssK and TssA in V. cholerae. Collectively, we demonstrate that chimeric VgrG-effector pairs could bypass the requirement of heterologous VgrG complex and propose that effector-stuffing inside the baseplate complex, facilitated by chaperones and the interaction with structural proteins, serves as a crucial structural determinant for T6SS assembly. Author summary: Effectors of bacterial secretion systems are generally considered as secreted proteins for interspecies interactions rather than components of the secretion apparatus. Our results reveal the complex interactions of effectors, chaperones, and structural proteins are crucial for T6SS assembly, suggesting an integral role of effectors as parts of the apparatus and distinctive from other secretion systems. [ABSTRACT FROM AUTHOR]

Paraburkholderia phymatum STM815, a rhizobial strain of the Burkholderiaceae family, is able to nodulate a broad range of legumes including the agriculturally important Phaseolus vulgaris (common bean). P. phymatum harbors two type VI Secretion Systems (T6SS-b and T6SS-3) in its genome that contribute to its high interbacterial competitiveness in vitro and in infecting the roots of several legumes. In this study, we show that P. phymatum T6SS-b is found in the genomes of several soil-dwelling plant symbionts and that its expression is induced by the presence of citrate and is higher at 20/28°C compared to 37°C. Conversely, T6SS-3 shows homologies to T6SS clusters found in several pathogenic Burkholderia strains, is more prominently expressed with succinate during stationary phase and at 37°C. In addition, T6SS-b expression was activated in the presence of germinated seeds as well as in P. vulgaris and Mimosa pudica root nodules. Phenotypic analysis of selected deletion mutant strains suggested a role of T6SS-b in motility but not at later stages of the interaction with legumes. In contrast, the T6SS-3 mutant was not affected in any of the free-living and symbiotic phenotypes examined. Thus, P. phymatum T6SS-b is potentially important for the early infection step in the symbiosis with legumes. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is a widespread mechanism of protein delivery into target cells, present in more than a quarter of all sequenced Gram-negative bacteria. The T6SS constitutes an important virulence factor, as it is responsible for targeting effectors in both prokaryotic and eukaryotic cells. The T6SS comprises a tail structure tethered to the cell envelope via a trans-envelope complex. In most T6SS, the membrane complex is anchored to the cell wall by the TagL accessory protein. In this study, we report the first crystal structure of a peptidoglycan-binding domain of TagL. The fold is conserved with members of the OmpA/Pal/MotB family, and more importantly, the peptidoglycan binding site is conserved. This structure further exemplifies how proteins involved in anchoring to the cell wall for different cellular functions rely on an interaction network with peptidoglycan strictly conserved. [ABSTRACT FROM AUTHOR]

Background: The type VI protein secretion system (T6SS) is important in diverse cellular processes in Gram-negative bacteria, including interactions with other bacteria and with eukaryotic hosts. In this study we analyze the evolution of the T6SS in the genus Xanthomonas and evaluate its importance of the T6SS for virulence and in vitro motility in Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of bacterial blight in cassava (Manihot esculenta). We delineate the organization of the T6SS gene clusters in Xanthomonas and then characterize proteins of this secretion system in Xpm strain CIO151. Results: We describe the presence of three different clusters in the genus Xanthomonas that vary in their organization and degree of synteny between species. Using a gene knockout strategy, we also found that vgrG and hcp are required for maximal aggressiveness of Xpm on cassava plants while clpV is important for both motility and maximal aggressiveness. Conclusion: We characterized the T6SS in 15 different strains in Xanthomonas and our phylogenetic analyses suggest that the T6SS might have been acquired by a very ancient event of horizontal gene transfer and maintained through evolution, hinting at their importance for the adaptation of Xanthomonas to their hosts. Finally, we demonstrated that the T6SS of Xpm is functional, and significantly contributes to virulence and motility. This is the first experimental study that demonstrates the role of the T6SS in the Xpm-cassava interaction and the T6SS organization in the genus Xanthomonas. [ABSTRACT FROM AUTHOR]

The health of mammals depends on a complex interplay with their microbial ecosystems. Compartments exposed to external environments such as the mucosal surfaces of the gastrointestinal tract accommodate the gut microbiota, composed by a wide range of bacteria. The gut microbiome confers benefits to the host, including expansion of metabolic potential and the development of an immune system that can robustly protect from external and internal insults. The cooperation between gut microbiome and host is enabled in part by the formation of partitioned niches that harbor diverse bacterial phyla. Bacterial secretion systems are commonly employed to manipulate the composition of these local environments. Here, we explore the roles of the bacterial type VI secretion system (T6SS), present in ~25% of gram-negative bacteria, including many symbionts, in the establishment and perturbation of bacterial commensalism, and symbiosis in host mucosal sites. This versatile apparatus drives bacterial competition, although in some cases can also interfere directly with host cells and facilitate nutrient acquisition. In addition, some bacterial pathogens cause disease when their T6SS leads to dysbiosis and subverts host immune responses in defined animal models. This review explores our knowledge of the T6SS in the context of the "host-microbiota-pathogen" triumvirate and examines contexts in which the importance of this secretion system may be underappreciated. [ABSTRACT FROM AUTHOR]

Summary: The type VI nanomachine is critical for Vibrio cholerae to establish infections and to thrive in niches co‐occupied by competing bacteria. The genes for the type VI structural proteins are encoded in one large and two small auxiliary gene clusters. VCA0117 (VasH) – a σ54‐transcriptional activator – is strictly required for functionality of the type VI secretion system since it controls production of the structural protein Hcp. While some strains constitutively produce a functional system, others do not and require specific growth conditions of low temperature and high osmolarity for expression of the type VI machinery. Here, we trace integration of these regulatory signals to the promoter activity of the large gene cluster in which many components of the machinery and VCA0117 itself are encoded. Using in vivo and in vitro assays and variants of VCA0117, we show that activation of the σ54‐promoters of the auxiliary gene clusters by elevated VCA0117 levels are all that is required to overcome the need for specialized growth conditions. We propose a model in which signal integration via the large operon promoter directs otherwise restrictive levels of VCA0117 that ultimately dictates a sufficient supply of Hcp for completion of a functional type VI secretion system. [ABSTRACT FROM AUTHOR]

Summary: With the rapid increase of aquaculture contributing to sustainable food security, comes the need to better understand seafood associated diseases. One of the major aquatic bacterial genera responsible for human infections from seafood is Vibrio, especially from oysters. Currently, in vivo study of bacterial interactions within oysters is limited by the inability to promote high‐level uptake of bacteria by oysters. This study has therefore evolved current natural marine snow protocols to generate 'artificial' marine snow, into which bacteria can be incorporated to facilitate extensive uptake by oysters. This presents an adaptable model for bacterial study within filter‐feeding shellfish. Using this model, we demonstrate for the first time the antibacterial activity of Vibrio vulnificus Type 6 secretion systems in vivo, revealing an important role for the T6SS in V. vulnificus ecology. [ABSTRACT FROM AUTHOR]

Summary: Pacific oyster mortality syndrome affects juveniles of Crassostrea gigas oysters and threatens the sustainability of commercial and natural stocks of this species. Vibrio crassostreae (V. crassostreae) has been repeatedly isolated from diseased animals, and the majority of the strains have been demonstrated to be virulent for oysters. In this study, we showed that oyster farms exhibited a high prevalence of a virulence plasmid carried by V. crassostreae, while oysters, at an adult stage, were reservoirs of this virulent population. The pathogenicity of V. crassostreae depends on a novel transcriptional regulator, which activates the bidirectional promoter of a type 6 secretion system (T6SS) genes cluster. Both the T6SS and a second chromosomal virulence factor, r5.7, are necessary for virulence but act independently to cause haemocyte (oyster immune cell) cytotoxicity. A phylogenetically closely related T6SS was identified in V. aestuarianus and V. tapetis, which infect adult oysters and clams respectively. We propose that haemocyte cytotoxicity is a lethality trait shared by a broad range of mollusc pathogens, and we speculate that T6SS was involved in parallel evolution of pathogen for molluscs. [ABSTRACT FROM AUTHOR]

Enterobacter cloacae has emerged as an opportunistic pathogen in healthcare-associated infections. Analysis of the genomic sequences of several E. cloacae strains revealed the presence of genes that code for expression of at least one type VI secretion system (T6SS). Here, we report that E. cloacae strain ATCC 13047 codes for two functional T6SS named T6SS-1 and T6SS-2. T6SS-1 and T6SS-2 were preferentially expressed in tryptic soy broth and tissue culture medium (DMEM), respectively. Mutants in T6SS-1-associated genes clpV1 and hcp1 significantly affected their ability of inter- and intra-bacterial killing indicating that T6SS-1 is required for bacterial competition. In addition, the Hcp effector protein was detected in supernatants of E. cloacae cultures and a functional T6SS-1 was required for the secretion of this protein. A clpV2 mutant was impaired in both biofilm formation and adherence to epithelial cells, supporting the notion that these phenotypes are T6SS-2 dependent. In vivo data strongly suggest that both T6SSs are required for intestinal colonization because single and double mutants in clpV1 and clpV2 genes were defective in gut colonization in mice. We conclude that the two T6SSs are involved in the pathogenesis scheme of E. cloacae with specialized functions in the interaction with other bacteria and with host cells. [ABSTRACT FROM AUTHOR]

The Type VI secretion system (T6SS) is a protein translocation nanomachine widespread among Gram‐negative bacteria and used as a means to deliver effectors directly into target bacterial or eukaryotic cells. These effectors have a wide variety of functions within target cells that ultimately help the secreting cell gain a competitive fitness advantage. Here, we discuss the different ways in which these effectors can be delivered by the T6SS and the diverse mechanisms by which they exert their noxious action upon recipient cells. We also highlight the existence of roles for T6SS effectors beyond simply the killing of neighbouring cells. [ABSTRACT FROM AUTHOR]

In the environment, bacteria show close association, such as interspecies interaction, with other bacteria as well as host organisms. The type VI secretion system (T6SS) in gram‐negative bacteria is involved in bacterial competition or virulence. The plant pathogen Burkholderia glumae BGR1, causing bacterial panicle blight in rice, has four T6SS gene clusters. The presence of at least one T6SS gene cluster in an organism indicates its distinct role, like in the bacterial and eukaryotic cell targeting system. In this study, deletion mutants targeting four tssD genes, which encode the main component of T6SS needle formation, were constructed to functionally dissect the four T6SSs in B. glumae BGR1. We found that both T6SS group_4 and group_5, belonging to the eukaryotic targeting system, act independently as bacterial virulence factors toward host plants. In contrast, T6SS group_1 is involved in bacterial competition by exerting antibacterial effects. The ΔtssD1 mutant lost the antibacterial effect of T6SS group_1. The ΔtssD1 mutant showed similar virulence as the wild‐type BGR1 in rice because the ΔtssD1 mutant, like the wild‐type BGR1, still has key virulence factors such as toxin production towards rice. However, metagenomic analysis showed different bacterial communities in rice infected with the ΔtssD1 mutant compared to wild‐type BGR1. In particular, the T6SS group_1 controls endophytic plant‐associated bacteria such as Luteibacter and Dyella in rice plants and may have an advantage in competing with endophytic plant‐associated bacteria for settlement inside rice plants in the environment. Thus, B. glumae BGR1 causes disease using T6SSs with functionally distinct roles. [ABSTRACT FROM AUTHOR]

The pathogenesis of the rice pathogenic bacterium Burkholderia glumae is under the tight regulation of the tofI/tofR quorum‐sensing (QS) system. tepR, encoding a group I bacterial enhancer‐binding protein, negatively regulates the production of toxoflavin, the phytotoxin acting as a major virulence factor in B. glumae. In this study, through a transcriptomic analysis, we identified the genes that were modulated by tepR and/or the tofI/tofR QS system. More than half of the differentially expressed genes, including the genes for the biosynthesis and transport of toxoflavin, were significantly more highly expressed in the ΔtepR mutant but less expressed in the ΔtofI‐tofR (tofI/tofR QS‐defective) mutant. In consonance with the transcriptome data, other virulence‐related functions of B. glumae, extracellular protease activity and flagellum‐dependent motility, were also negatively regulated by tepR, and this negative regulatory function of tepR was dependent on the IclR‐type transcriptional regulator gene qsmR. Likewise, the ΔtepR mutant exhibited a higher level of heat tolerance in congruence with the higher transcription levels of heat shock protein genes in the mutant. Interestingly, tepR also exhibited its positive regulatory function on a previously uncharacterized type VI secretion system (denoted as BgT6SS‐1). The survival of the both ΔtepR and ΔtssD (BgT6SS‐1‐defective) mutants was significantly compromised compared to the wild‐type parent strain 336gr‐1 in the presence of the natural rice‐inhabiting bacterium, Pantoea sp. RSPAM1. Taken together, this study revealed pivotal regulatory roles of tepR in orchestrating multiple biological functions of B. glumae, including pathogenesis, heat tolerance, and bacterial interspecies competition. [ABSTRACT FROM AUTHOR]

Ralstonia solanacearum is an important soil-borne plant pathogen which causes bacterial wilt in a large number of crops. Bacterial Type Six Secretion System (T6SS) is known to participate in pathogenesis, bacterial interaction and inter-bacterial competition. Contribution of T6SS in the virulence of R. solanacearum on eggplant (Solanum melongena L) is studied. In this study, five T6SS gene (ompA, vgrG3, hcp, tssH and tssM) mutants have been developed by insertional mutagenesis and the virulence of the mutants was evaluated on eggplant. In general, the T6SS mutants showed significant reduction of wilt on eggplant. R. solanacearum mutant of ompA gene significantly reduced the wilt from day five through day eight in petiole inoculation. In soil drench inoculation, R. solanacearum mutant of vgrG3 gene reduced the wilt on eggplant and was significantly different throughout the experimental period. Other mutants, viz., tssH, tssM and hcp, also reduced the wilt during the initial stages of disease development. This is the first report on the role of T6SS genes, ompA, vgrG3, hcp and tssH on virulence of R. solanacearum. [ABSTRACT FROM AUTHOR]

Vibrios, which include more than 120 valid species, are an abundant and diverse group of bacteria in marine and estuarine environments. Some of these bacteria have been recognized as pathogens of both marine animals and humans, and therefore, their virulence mechanisms have attracted increasing attention. The type III secretion system (T3SS) is an important virulence determinant in many gram-negative bacteria, in which this system directly translocates variable effectors into the host cytosol for the manipulation of the cellular responses. In this study, the distribution of the T3SS gene cluster was first examined in 110 Vibrio strains of 26 different species, including 98 strains isolated from coastal areas in China. Several T3SS1 genes, but not T3SS2 genes (T3SS2α and T3SS2β), were universally detected in all the strains of four species, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi , and Vibrio campbellii. The effector coding regions within the T3SS1 gene clusters from the T3SS1-positive strains were further analyzed, revealing that variations in the effectors of Vibrio T3SS1 were observed among the four Vibrio species, even between different strains in V. harveyi , according to their genetic organization. Importantly, Afp17, a potential novel effector that may exert a similar function as the known effector VopS in T3SS1-induced cell death, based on cytotoxicity assay results, was found in the effector coding region of the T3SS1 in some V. harveyi and V. campbellii strains. Finally, it was revealed that differences in T3SS1-mediated cytotoxicity were dependent not only on the variations in the effectors of Vibrio T3SS1 but also on the initial adhesion ability to host cells, which is another prerequisite condition. Altogether, our results contribute to the clarification of the diversity of T3SS1 effectors and a better understanding of the differences in cytotoxicity among Vibrio species. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is a widespread bacterial nanoweapon used for delivery of toxic proteins into cell targets and contributes to virulence, anti-inflammatory processes, and interbacterial competition. In the model phytopathogenic bacterium Pseudomonas syringae pv. tomato (Pst) DC3000, two T6SS gene clusters, HSI-I and HSI-II, were identified, but their functions remain unclear. We previously reported that hcp2 , located in HSI-II, is involved in competition with enterobacteria and yeast. Here, we demonstrated that interbacterial competition of Pst DC3000 against several Gram-negative plant-associated bacteria requires mainly HSI-II activity. By means of a systematic approach using in-frame deletion mutants for each gene in the HSI-II cluster, we identified genes indispensable for Hcp2 expression, Hcp2 secretion and interbacterial competition ability. Deletion of PSPTO_5413 only affected growth in interbacterial competition assays but not Hcp2 secretion, which suggests that PSPTO_5413 might be a putative effector. Moreover, PSPTO_5424, encoding a putative σ54-dependent transcriptional regulator, positively regulated the expression of all three operons in HSI-II. Our discovery that the HSI-II gene cluster gives Pst DC3000 the ability to compete with other plant-associated bacteria could help in understanding a possible mechanism of how phytopathogenic bacteria maintain their ecological niches. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is a multiprotein weapon that kills eukaryotic predators or prokaryotic competitors by delivering toxic effectors. Despite the importance of T6SS in bacterial environmental adaptation, it is still challenging to systematically identify T6SS effectors because of their high diversity and lack of conserved domains. In this report, we discovered a putative effector gene, U876-17730, in the whole genome of Aeromonas hydrophila NJ-35 based on the reported conservative domain DUF4123 (domain of unknown function), with two cognate immunity proteins encoded downstream. Phylogenetic tree analysis of amino acids indicates that AH17730 belongs to the Tle1 (type VI lipase effector) family, and therefore was named Tle1AH. The deletion of tle1AH resulted in significantly decreased biofilm formation, antibacterial competition ability and virulence in zebrafish (Danio rerio) when compared to the wild-type strain. Only when the two immunity proteins coexist can bacteria protect themselves from the toxicity of Tle1AH. Further study shows that Tle1AH is a kind of phospholipase that possesses a conserved lipase motif, Gly-X-Ser-X-Gly (X is for any amino acid). Tle1AH is secreted by T6SS, and this secretion requires its interaction with an associated VgrG (valine-glycine repeat protein G). In conclusion, we identified a T6SS effector-immunity pair and verified its function, which lays the foundation for future research on the role of T6SS in the pathogenic mechanism of A. hydrophila. [ABSTRACT FROM AUTHOR]

Bacterial Rhs proteins containing toxic domains are often secreted by type VI secretion systems (T6SSs) through unclear mechanisms. Here, we show that the T6SS Rhs-family effector TseI of Aeromonas dhakensis is subject to self-cleavage at both the N- and the C-terminus, releasing the middle Rhs core and two VgrG-interacting domains (which we name VIRN and VIRC). VIRC is an endonuclease, and the immunity protein TsiI protects against VIRC toxicity through direct interaction. Proteolytic release of VIRC and VIRN is mediated, respectively, by an internal aspartic protease activity and by two conserved glutamic residues in the Rhs core. Mutations abolishing self-cleavage do not block secretion, but reduce TseI toxicity. Deletion of VIRN or the Rhs core abolishes secretion. TseI homologs from Pseudomonas syringae, P. aeruginosa, and Vibrio parahaemolyticus are also self-cleaved. VIRN and VIRC interact with protein VgrG1, while the Rhs core interacts with protein TecI. We propose that VIRN and the Rhs core act as T6SS intramolecular chaperones to facilitate toxin secretion and function. Bacterial Rhs proteins with toxic domains are often secreted by type VI secretion systems. Here, the authors show that one of these proteins self-cleaves into three fragments, with the Rhs core and the N-terminal domain facilitating secretion and function of the C-terminal toxic domain. [ABSTRACT FROM AUTHOR]

Background: The inner membrane protein DotU of Aeromonas veronii is an important component of the minimal core conserved membrane proteome required for the formation of an envelope-transmembrane complex. This protein functions in a type VI secretion system (T6SS), and the role of this T6SS during the pathogenic process has not been clearly described. Results: A recombinant A. veronii with a partial disruption of the dotU gene (720 bp of the in-frame sequence) (defined as ∆dotU) was constructed by two conjugate exchanges. We found that the mutant ∆dotU allele can be stably inherited for more than 50 generations. Inactivation of the A. veronii dotU gene resulted in no significant changes in growth or resistance to various environmental changes. However, compared with the wild-type strain colony, the mutant ∆dotU colony had a rough surface morphology. In addition, the biofilm formation ability of the mutant ∆dotU was significantly enhanced by 2.1-fold. Conversely, the deletion of the dotU gene resulted in a significant decrease in pathogenicity and infectivity compared to those of the A. veronii wild-type strain. Conclusions: Our findings indicated that the dotU gene was an essential participant in the pathogenicity and invasiveness of A. veronii TH0426, which provides a novel perspective on the pathogenesis of TH0426 and lays the foundation for discovering potential T6SS effectors. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possesses an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition and identified VgrG4 as a T6SS effector. The DUF2345 domain of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition. Author summary: Klebsiella pneumoniae has been singled out as an "urgent threat to human health" due to extremely drug resistant strains. Numerous studies investigate the molecular mechanisms underlying antibiotic resistance in K. pneumoniae, while others dissect the virulence strategies of this pathogen. However, there is still limited knowledge on the fitness of Klebsiella in the environment, and, particularly, the competition of Klebsiella with other species. Here, we demonstrate that Klebsiella exploits the type VI secretion system (T6SS) nanoweapon to kill bacterial competitors and fungi. K. pneumoniae perceives T6SS attacks from bacterial competitors, resulting in retaliation against the aggressive cell. The perception of the attack involved the sensor PhoPQ and led to the up-regulation of the T6SS. We identified one of the toxins deployed by the T6SS to antagonize other microbes and revealed how Klebsiella protects itself from this toxin. Our findings provide a better understanding of the T6SS role in microbial competition and uncover new aspects on how bacteria regulate T6SS-mediated microbial antagonism. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is a contractile injection apparatus that translocates a spike loaded with various effectors directly into eukaryotic or prokaryotic target cells. Pseudomonas aeruginosa can load either one of its three T6SSs with a variety of toxic bullets using different but specific modes. The T6SS spike, which punctures the bacterial cell envelope allowing effector transport, consists of a torch-like VgrG trimer on which sits a PAAR protein sharpening the VgrG tip. VgrG itself sits on the Hcp tube and all elements, packed into a T6SS sheath, are propelled out of the cell and into target cells. On occasion, effectors are covalent extensions of VgrG, PAAR or Hcp proteins, which are then coined "evolved" components as opposed to canonical. Here, we show how various passenger domains could be fused to the C terminus of a canonical VgrG, VgrG1a from P. aeruginosa, and be sent into the bacterial culture supernatant. There is no restriction on the passenger type, although the efficacy may vary greatly, since we used either an unrelated T6SS protein, β-lactamase, a covalent extension of an "evolved" VgrG, VgrG2b, or a Hcp-dependent T6SS toxin, Tse2. Our data further highlights an exceptional modularity/flexibility for loading the T6SS nano-weapon. Refining the parameters to optimize delivery of passenger proteins of interest would have attractive medical and industrial applications. This may for example involve engineering the T6SS as a delivery system to shuttle toxins into either bacterial pathogens or tumour cells which would be an original approach in the fight against antimicrobial resistant bacteria or cancer. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Many Gram-negative bacteria use a type VI secretion system (T6SS) for microbial warfare and/or host manipulation. Acinetobacter baumannii is an important nosocomial pathogen and many A. baumannii strains utilize a T6SS to deliver toxic effector proteins to surrounding bacterial cells. These toxic effectors are usually delivered together with VgrG proteins, which form part of the T6SS tip complex. All previously identified A. baumannii T6SS effectors are encoded within a three- or four-gene locus that also encodes a cognate VgrG and immunity protein, and sometimes a chaperone. In order to characterize the diversity and distribution of T6SS effectors and immunity proteins in this species, we first identified all vgrG genes in 97 A. baumannii strains via the presence of the highly conserved VgrG domain. Most strains encoded between two and four different VgrG proteins. We then analyzed the regions downstream of the identified vgrG genes and identified more than 240 putative effectors. The presence of conserved domains in these effectors suggested a range of functions, including peptidoglycan hydrolases, lipases, nucleases, and nucleic acid deaminases. However, 10 of the effector groups had no functionally characterized domains. Phylogenetic analysis of these putative effectors revealed that they clustered into 32 distinct groups that appear to have been acquired from a diverse set of ancestors. Corresponding immunity proteins were identified for all but two of the effector groups. Effectors from eight of the 32 groups contained N-terminal rearrangement hotspot (RHS) domains. The C-terminal regions of these RHS proteins, which are predicted to confer the toxic effector function, were very diverse, but the N-terminal RHS domains clustered into just two groups. While the majority of A. baumannii strains contained an RHS type effector, no strains encoded two RHS effectors with similar N-terminal sequences, suggesting that the presence of similar N-terminal RHS domains leads to competitive exclusion. Together, these analyses define the extreme diversity of T6SS effectors within A. baumannii and, as many have unknown functions, future detailed characterization of these effectors may lead to the identification of proteins with novel antibacterial properties. [ABSTRACT FROM AUTHOR]

The complexity of plant microbial communities provides a rich model for investigating biochemical and regulatory strategies involved in interbacterial competition. Within these niches, the soft rot Enterobacteriaceae (SRE) represents an emerging group of plant–pathogens causing soft rot/blackleg diseases resulting in economic losses worldwide in a variety of crops. A preliminary screening using next-generation sequencing of 16S rRNA comparatively analyzing healthy and diseased potato tubers, identified several taxa from Proteobacteria to Firmicutes as potential potato endophytes/plant pathogens. Subsequent to this, a range of molecular and computational techniques were used to determine the contribution of antimicrobial factors such as bacteriocins, carbapenem and type VI secretion system (T6SS), found in an aggressive SRE (Pectobacterium carotovorum subsp. brasiliense strain PBR1692 – Pcb 1692) against these endophytes/plant pathogens. The results showed growth inhibition of several Proteobacteria by Pcb 1692 depends either on carbapenem or pyocin production. Whereas for targeted Firmicutes , only the Pcb 1692 pyocin seems to play a role in growth inhibition. Furthermore, production of carbapenem by Pcb 1692 was observably dependent on the presence of environmental iron and oxygen. Additionally, upon deletion of fur , sly A and exp I regulators, carbapenem production ceased, implying a complex regulatory mechanism involving these three genes. Finally, the results demonstrated that although T6SS confers no relevant advantage during in vitro competition, a significant attenuation in competition by the mutant strain lacking a functional T6SS was observed in planta. IMPORTANCE: Soft rot Enterobacteriaceae (SRE) represents important phytopathogens causing soft rot/blackleg diseases in a variety of crops leading to huge economic losses worldwide. These pathogens have been isolated alongside other bacteria from different environments such as potato tubers, stems, roots and from the soil. In these environments, SREs coexist with other bacteria where they have to compete for scarce nutrients and other resources. In this report, we show that Pectobacterium carotovorum subsp. brasiliense strain PBR1692 – Pcb 1692, which represents one of the SREs, inhibits growth of several different bacteria by producing different antimicrobial compounds. These antimicrobial compounds can be secreted inside or outside the plant host, allowing Pcb 1692 to effectively colonize different types of ecological niches. By analyzing the genome sequences of several SREs, we show that other SREs likely deploy similar antimicrobials to target other bacteria. [ABSTRACT FROM AUTHOR]

Bacterial pathogens utilize a myriad of mechanisms to invade mammalian hosts, damage tissue sites, and evade the immune system. One essential strategy of Gram-negative bacteria is the secretion of virulence factors through both inner and outer membranes to reach a potential target. Most secretion systems are harbored in mobile elements including transposons, plasmids, pathogenicity islands, and phages, and Escherichia coli is one of the more versatile bacteria adopting this genetic information by horizontal gene transfer. Additionally, E. coli is a bacterial species with members of the commensal intestinal microbiota and pathogens associated with numerous types of infections such as intestinal, urinary, and systemic in humans and other animals. T6SS cluster plasticity suggests evolutionarily divergent systems were acquired horizontally. T6SS is a secretion nanomachine that is extended through the bacterial double membrane; from this apparatus, substrates are conveyed straight from the cytoplasm of the bacterium into a target cell or to the extracellular space. This nanomachine consists of three main complexes: proteins in the inner membrane that are T4SS component-like, the baseplate complex, and the tail complex, which are formed by components evolutionarily related to contractile bacteriophage tails. Advances in the T6SS understanding include the functional and structural characterization of at least 13 subunits (so-called core components), which are thought to comprise the minimal apparatus. So far, the main role of T6SS is on bacterial competition by using it to kill neighboring non-immune bacteria for which antibacterial proteins are secreted directly into the periplasm of the bacterial target after cell–cell contact. Interestingly, a few T6SSs have been associated directly to pathogenesis, e.g., roles in biofilm formation and macrophage survival. Here, we focus on the advances on T6SS from the perspective of E. coli pathotypes with emphasis in the secretion apparatus architecture, the mechanisms of pathogenicity of effector proteins, and the events of lateral gene transfer that led to its spread. [ABSTRACT FROM AUTHOR]

The bacterial pathogen Pseudomonas aeruginosa uses three type VI secretion systems (T6SSs) to drive a multitude of effector proteins into eukaryotic or prokaryotic target cells. The T6SS is a supramolecular nanomachine, involving a set of 13 core proteins, which resembles the contractile tail of bacteriophages and whose tip is considered as a puncturing device helping to cross membranes. Effectors can attach directly to the T6SS spike which is composed of a VgrG (valine-glycine-rich proteins) trimer, of which P. aeruginosa produces several. We have previously shown that the master regulator RsmA controls the expression of all three T6SS gene clusters (H1-, H2- and H3-T6SS) and a range of remote vgrG and effector genes. We also demonstrated that specific interactions between VgrGs and various T6SS effectors are prerequisite for effector delivery in a process we called "à la carte delivery." Here, we provide an in-depth description on how the two H2-T6SS-dependent effectors PldA and PldB are delivered via their cognate VgrGs, VgrG4b and VgrG5, respectively. We show that specific recognition of the VgrG C terminus is required and effector specificity can be swapped by exchanging these C-terminal domains. Importantly, we established that effector recognition by a cognate VgrG is not always sufficient to achieve successful secretion, but it is crucial to provide effector stability. This study highlights the complexity of effector adaptation to the T6SS nanomachine and shows how the VgrG tip can possibly be manipulated to achieve effector delivery. [ABSTRACT FROM AUTHOR]