The VgrG Proteins Are "à la Carte" Delivery Systems for Bacterial Type VI Effectors

The bacterial type VI secretion system (T6SS) is a supra-molecular complex akin to bacteriophage tails, with VgrG proteins acting as a puncturing device. The Pseudomonas aeruginosa H1-T6SS has been extensively characterized. It is involved in bacterial killing and in the delivery of three toxins, Tse1–3. Here, we demonstrate the independent contribution of the three H1-T6SS co-regulated vgrG genes, vgrG1abc, to bacterial killing. A putative toxin is encoded in the vicinity of each vgrG gene, supporting the concept of specific VgrG/toxin couples. In this respect, VgrG1c is involved in the delivery of an Rhs protein, RhsP1. The RhsP1 C terminus carries a toxic activity, from which the producing bacterium is protected by a cognate immunity. Similarly, VgrG1a-dependent toxicity is associated with the PA0093 gene encoding a two-domain protein with a putative toxin domain (Toxin_61) at the C terminus. Finally, VgrG1b-dependent killing is detectable upon complementation of a triple vgrG1abc mutant. The VgrG1b-dependent killing is mediated by PA0099, which presents the characteristics of the superfamily nuclease 2 toxin members. Overall, these data develop the concept that VgrGs are indispensable components for the specific delivery of effectors. Several additional vgrG genes are encoded on the P. aeruginosa genome and are not linked genetically to other T6SS genes. A closer inspection of these clusters reveals that they also encode putative toxins. Overall, these associations further support the notion of an original form of secretion system, in which VgrG acts as the carrier

The rise of the Type VI secretion system

Bacterial cells have developed multiple strategies to communicate with their surrounding environment. The intracellular compartment is separated from the milieu by a relatively impermeable cell envelope through which small molecules can passively diffuse, while larger macromolecules, such as proteins, can be actively transported. In Gram-negative bacteria, the cell envelope is a double membrane, which houses several supramolecular protein complexes that facilitate the trafficking of molecules. For example, bacterial pathogens use these types of machines to deliver toxins into target eukaryotic host cells, thus subverting host cellular functions. Six different types of nanomachines, called Type I - Type VI secretion systems (T1SS - T6SS), can be readily identified by their composition and mode of action. A remarkable feature of these protein secretion systems is their similarity to systems with other biological functions, such as motility or the exchange of genetic material. The T6SS has provided a refreshing view on this concept since it shares similarity with the puncturing device of bacteriophages, which is used by these viruses to inject their DNA into bacterial target cells. In contrast, the bacterial T6SS transports toxins into other bacteria, engaging a ferocious competition for the colonization of their environment. Moreover, as with few other secretion systems, the T6SS is capable of injecting toxins into eukaryotic cells, which contributes to a successful infection. This highlights the multifunctional aspects of the T6SS, and our understanding of its mechanistic details is an intense field of investigation with significant implications for ecology, agriculture and medicine

Comparative genomics of type VI secretion systems in strains of Pantoea ananatis from different environments

BACKGROUND: The Type VI secretion system (T6SS) has been identified in several different bacteria, including the plant pathogenPantoea ananatis. Previous in silico analyses described three different T6SS loci present in the pathogenic strain of P. ananatis LMG 20103. This initial investigation has been extended to include an additional seven sequenced strains of P. ananatis together with 39 strains from different ecological niches. Comparative and phylogenetic analyses were used to investigate the distribution, evolution, intra-strain variability and operon structure of the T6SS in the sequenced strains. RESULTS: Three different T6SS loci were identified in P. ananatis strain LMG 20103 and designated PA T6SS 1-3. PA T6SS-1 was present in all sequenced strains of P. ananatis and in all 39 additional strains examined in this study. In addition, PA T6SS-1 included all 13 core T6SS genes required for synthesis of a functional T6SS. The plasmid-borne PA T6SS-2 also included all 13 core T6SS genes but was restricted to only 33% (15/46) of the strains examined. In addition, PA T6SS-2 was restricted to strains of P. ananatis isolated from symptomatic plant material. This finding raises the possibility of an association between PA T6SS-2 and either pathogenicity or host specificity. The third cluster PA T6SS-3 was present in all strains analyzed in this study but lacked 11 of the 13 core T6SS genes suggesting it may not encoded a functional T6SS. Inter-strain variability was also associated with hcp and vgrG islands, which are associated with the T6SS and encode a variable number of proteins usually of unknown function. These proteins may play a role in the fitness of different strains in a variety of ecological niches or as candidate T6SS effectors. Phylogenetic analysis indicated that PA T6SS-1 and PA T6SS-2 are evolutionarily distinct. CONCLUSION: Our analysis indicates that the three T6SSs of P. ananatis appear to have been independently acquired and may play different roles relating to pathogenicity, host range determination and/or niche adaptation. Future work will be directed toward understanding the roles that these T6SSs play in the biology of P. ananatis.The University of Pretoria, the National Research Foundation (NRF), the Forestry and Agricultural Biotechnology Institute (FABI), the Tree Protection Co-operative Programme (TPCP), the NRF/Department of Science and Technology Centre of Excellence in Tree Health Biotechnology (CTHB), and the THRIP support program of the Department of Trade and Industry, South Africa.http://www.biomedcentral.com/bmcgenomics/am201

Comparative genomics reveals metabolic specificity of endozoicomonas isolated from a marine sponge and the genomic repertoire for host-bacteria symbioses

The most recently described bacterial members of the genus Endozoicomonas have been found in association with a wide variety of marine invertebrates. Despite their ubiquity in the host holobiont, limited information is available on the molecular genomic signatures of the symbiotic association of Endozoicomonas with marine sponges. Here, we generated a draft genome of Endozoicomonas sp. OPT23 isolated from the intertidal marine sponge Ophlitaspongia papilla and performed comprehensive comparative genomics analyses. Genome-specific analysis and metabolic pathway comparison of the members of the genus Endozoicomonas revealed the presence of gene clusters encoding for unique metabolic features, such as the utilization of carbon sources through lactate, L-rhamnose metabolism, and a phenylacetic acid degradation pathway in Endozoicomonas sp. OPT23. Moreover, the genome harbors genes encoding for eukaryotic-like proteins, such as ankyrin repeats, tetratricopeptide repeats, and Sel1 repeats, which likely facilitate sponge-bacterium attachment. The genome also encodes major secretion systems and homologs of effector molecules that seem to enable the sponge-associated bacterium to interact with the sponge and deliver the virulence factors for successful colonization. In conclusion, the genome analysis of Endozoicomonas sp. OPT23 revealed the presence of adaptive genomic signatures that might favor their symbiotic lifestyle within the sponge host.Anoop Alex was supported in part by the project PTDC/BIA-BMA/29985/2017 (POCI-01-0145-FEDER- 029985) from the European Regional Development Fund (ERDF) through COMPETE 2020—Operational Program for Competitiveness and Internationalization (POCI) and National Funds through the Fundação para a Ciência e a Tecnologia (FCT)/MCTES. Agostinho Antunes was funded in part by the Strategic Funding UID/Multi/04423/2019 through National Funds provided by FCT and the ERDF in the framework of the program PT2020, by the European Structural and Investment Funds (ESIF) through the Competitiveness and Internationalization Operational Program - COMPETE 2020 and by National Funds through the FCT under the project PTDC/AAG-GLO/6887/2014 (POCI-01-0124-FEDER-016845)

Genetic dissection of the type VI secretion system in Acinetobacter and identification of a novel peptidoglycan hydrolase, TagX, required for its biogenesis

The type VI secretion system (T6SS) is a widespread secretory apparatus produced by Gram-negative bacteria that has emerged as a potent mediator of antibacterial activity during interbacterial interactions. Most Acinetobacter species produce a genetically conserved T6SS, although the expression and functionality of this system vary among different strains. Some pathogenic Acinetobacter baumannii strains activate this secretion system via the spontaneous loss of a plasmid carrying T6SS repressors. In this work, we compared the expression of T6SS-related genes via transcriptome sequencing and differential proteomics in cells with and without the plasmid. This approach, together with the mutational analysis of the T6SS clusters, led to the determination of the genetic components required to elaborate a functional T6SS in the nosocomial pathogen A. baumannii and the nonpathogen A. baylyi. By constructing a comprehensive combination of mutants with changes in the T6SS-associated vgrG genes, we delineated their relative contributions to T6SS function. We further determined the importance of two effectors, including an effector-immunity pair, for antibacterial activity. Our genetic analysis led to the identification of an essential membrane-associated structural component named TagX, which we have characterized as a peptidoglycan hydrolase possessing l,d-endopeptidase activity. TagX shows homology to known bacteriophage l,d-endopeptidases and is conserved in the T6SS clusters of several bacterial species. We propose that TagX is the first identified enzyme that fulfills the important role of enabling the transit of T6SS machinery across the peptidoglycan layer of the T6SS-producing bacterium

Characterisation of the type VI secretion system in Pantoea ananatis

Pantoea ananatis is an important plant pathogen that causes disease symptoms in different plants worldwide. However, most of the virulence determinants of this pathogen have not been identified and functionally characterized. A previous study identified the type VI secretion system (T6SS) as a putative virulence determinant of P. ananatis strain LMG 20103, based on in silico analysis. This secretion system has been shown to play different roles in bacteria, including virulence, fitness and interbacterial competition. Therefore, the overall objective of this study was to determine the biological role (s) of the T6SS of P. ananatis. The first chapter of this thesis is a review of the literature, dealing with the different secretion systems used by Gram-negative bacteria to secrete effectors (toxins and proteins) from the cytoplasm to the exterior of the cell. Six different secretion systems have been identified in Gram-negative bacteria, i.e. T1SS to T6SS. These secretion systems have been functionally characterized and shown to play different roles related to virulence, fitness and inter-bacterial interactions. The T6SS represents the most recently described secretion system found in Gram-negative bacteria. Gene clusters encoding the T6SS are widespread in several pathogenic and non-pathogenic bacteria, with up to six genetically distinct T6SS gene clusters found in some bacterial species. This secretion system has been associated with different processes such as virulence, fitness, biofilm formation, niche colonization, and inter-bacterial competition. The T6SS can target cytotoxic effectors into either eukaryotes, prokaryotes or both. Genes encoding bactericidal and bacteriostatic effectors have been identified in the T6SS gene clusters of different bacteria. However, only a few of these effectors such as Hcp, VgrG, VasX, Tse, Tae, Tge, Tle, Ssp and Rhs toxins have been functionally characterized. In Chapter 2, a comparative analysis of the different T6SS gene clusters found in P. ananatis was undertaken. The T6SS-1 and T6SS-3 gene clusters of LMG 20103 were found to be conserved and syntenic in eight strains of P. ananatis of which genome sequences have been determined. Using PCR and probes, we also identified homologs of genes found in the T6SS-1 and T6SS-3 gene clusters in all 36 additional strains of P. ananatis of which the genome sequences have not been determined. The third cluster, T6SS-2 was found to be restricted to only three out of eight sequenced strains of P. ananatis, which included LMG 20103 (a pathogen of Eucalyptus spp.), PA-4 (a rice pathogen) and AJ13355 (a non-pathogenic strain isolated from the soil). Furthermore, T6SS-3 gene homologs were also identified in 12 out of 36 (33%) environmental strains of P. ananatis analyzed in this study. In Chapter 3, we functionally characterized the different T6SS gene clusters found in strains LMG 20103 and LMG 2665T. Our results indicated that the T6SS-1 of LMG 2665T plays a role in onion pathogenicity and growth inhibition of other bacteria. We also showed that the homologous T6SS-1 of strain LMG 20103 played a role in bacterial competition but was not required for pathogenicity in onion plants. Based on our assay conditions, no discernable phenotype was observed following deletion of the T6SS-2 and T6SS-3 gene clusters found in the genome sequences of either strains of P. ananatis. In Chapter 4, we carried out a genetic analysis of the tssA and tssD genes found in the T6SS-1 of strain LMG 2665T. This was done in part to validate results from Chapter 3, because the whole cluster deletion mutants were not complemented and secondly, to determine if these genes were required for T6S. Deletion of these genes abrogated pathogenicity in onion plants compared to the wild-type. In addition, the ΔtssA and ΔtssD mutants of strain LMG 2665T were unable to inhibit growth of other Gramnegative bacteria following co-culture on LB agar. In trans-expression of the fulllength tssA and tssD genes on a plasmid restored pathogenicity and inter-bacterial competition of the complemented T6SS mutants to near wild-type levels. These results, for the first time, demonstrated that the tssA and tssD genes of strain LMG 2665T are required for pathogenicity and inter-bacterial competition. We hypothesize that these genes encode proteins that are essential for the biosynthesis of a functional T6SS.Thesis (PhD)--University of Pretoria, 2014.Microbiology and Plant PathologyPhDUnrestricte

Investigation into the effector repertoire of the H2 type VI secretion system of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen, causing both acute and chronic infections. This bacterium displays remarkable adaptability and potential for virulence, partly due to its arsenal of protein secretion systems. The type VI secretion system (T6SS) is a contractile injection apparatus, firing a spear-like structure into target cells to deliver its cargo of effector proteins. P. aeruginosa encodes three such systems, denoted H1-, H2- and H3-T6SS. This dissertation discloses work focused on progressing our understanding of the H2-T6SS in this pathogen. We reveal that the H2-T6SS is controlled by the Gac/Rsm pathway, a major regulatory network in this pathogen responsible for the lifestyle switch between motile and sessile bacteria. Quorum sensing, the sophisticated signalling network governing social behaviour, is responsible for the expression of this secretion system in a growth-phase dependent manner, while temperature also has an input in a strain-dependent fashion. We advance our understanding of the composition of the H2-T6SS nanomachine, identifying multiple components of the spear-like delivery device, comprising an Hcp tube capped with a spike structure composed of three VgrGs and one PAAR protein. Importantly, we begin to decipher the payload of this secretion system, describing several phospholipase family effectors which confer a significant advantage to P. aeruginosa during bacterial competition. Building upon this, we propose a hierarchy of effector delivery determined by the VgrG/PAAR composition of the spike. Finally, we characterise a specific H2-T6SS effector: the C-terminal extension of the VgrG2b spike protein. Although we initially investigate its reported role within eukaryotic cells, we determine that this metallopeptidase-like effector is part of a wider antibacterial T6SS toxin family. We describe its cognate periplasmic immunity determinant and progress the elucidation of the target of the effector. Overall, we advance our understanding of the H2-T6SS of P. aeruginosa in terms of its regulation, organisation and cargo.Open Acces

Activation and functional studies of the Type VI secretion systems in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a versatile and prevalent opportunistic pathogen. It encodes a large arsenal of pathogenicity factors, and secrets a plethora of proteins using specialised protein secretion systems. The type VI secretion system (T6SS) delivers proteins directly into neighbouring bacteria or eukaryotic cells using a mechanism homologous to the T4 bacteriophage tail spike. Three T6SS are encoded on the P. aeruginosa genome. The study of the H1-T6SS has been facilitated by the fact it can be activated by the manipulation of the RetS/Gac/Rsm regulatory cascade by deletion of retS. However, the precise signals required for activation of this cascade, resulting in H1-T6SS activation, are unknown. This work investigates the role of subinhibitory concentrations of antibiotics in activating the system, and shows that kanamycin is able to induce production of core H1-T6SS components. This activation requires a functional Gac/Rsm cascade, but it is not known if this is due to direct signalling via the cascade, or due to a dominant effect of RsmA repression. The H2-T6SS is characterized in this work. We highlight key differences between the H2-T6SS cluster in PAO1 and PA14, including the presence of additional core T6SS components and putative secreted effectors. A strain is generated in which expression of the PA14 H2-T6SS cluster can be activated and tightly controlled by arabinose inducible promoters. The activity of the promoters is confirmed by the H2-T6SS dependent secretion of Hcp2 specifically upon arabinose induction. We further consider two putative H2-T6SS secreted substrates, VgrG14 and Rhs14. Production of these proteins is observed following arabinose induction, but their secretion is not detected. The Rhs14 protein is characterised, and its possible role as a H2-T6SS dependent effector is discussed. Finally, the H2-T6SS system in PA14 is shown to inhibit the internalisation of P. aeruginosa PA14, in contrast to the previously published observations of the H2-T6SS promoting internalisation of PAO1.Open Acces

Characterising the role of Vibrio vulnificus type 6 secretion systems 1 and 2 in an in vivo oyster model

Vibrio vulnificus is a significant human pathogen commonly isolated from temperate marine environments, where it is particularly abundant within filter-feeding shellfish. V. vulnificus is currently increasing in prevalence, theorised to be due to climate change facilitating V. vulnificus growth in previously inhospitable environments. Infection of susceptible individuals with V. vulnificus typically results in either primary septicaemia or necrotic wound infection, depending upon the route of entry, and frequently results in death if not treated rapidly. Two type 6 secretion systems (T6SS) have been identified in V. vulnificus, termed the T6SS1 and the T6SS2. The T6SS is a molecular syringe utilised to inject cytotoxic effector proteins into neighbouring cells. Whilst the T6SS2 is present in all sequenced V. vulnificus strains, only a subset possesses the T6SS1. Previous bacterial co-culture killing assays between T6SS1+ and T6SS1- V. vulnificus strains demonstrated thermoregulated T6SS1-mediated killing of T6SS1- strains. This study further characterised the role of both the T6SS1 and the T6SS2 in vitro. In vitro co-culture assays demonstrated that both the T6SS1 and the T6SS2 have antibacterial killing activity at the environmentally representative temperature of 21 °C. This is the first characterised role for the T6SS2 of V. vulnificus. No anti-eukaryotic activity was observed following co-culture with the phagocytic amoeba, Dictyostelium discoideum, suggesting that T6SS activity is purely antibacterial. In vitro bacterial co-culture assays were replicated in vivo using an oyster model. To facilitate high-level uptake of bacterial strains of interest by oysters, an artificial marine snow model was developed where bacteria were incorporated into easily ingested phytoplankton aggregates. Uptake of bacteria from artificial marine snow was extremely successful, resulting in bacterial loads within oysters significantly greater than achieved by any study to date. Using this model, this study was able to demonstrate that V. vulnificus utilises both the T6SS1 and the T6SS2 to target and kill neighbouring bacteria, in both an intra and inter-species manner. This data suggests that the T6SSs of V. vulnificus play a key role in V. vulnificus ecology and the dynamics between bacterial populations in vivo

Activation and functional studies of the Type VI secretion systems in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a versatile and prevalent opportunistic pathogen. It encodes a large arsenal of pathogenicity factors, and secrets a plethora of proteins using specialised protein secretion systems. The type VI secretion system (T6SS) delivers proteins directly into neighbouring bacteria or eukaryotic cells using a mechanism homologous to the T4 bacteriophage tail spike. Three T6SS are encoded on the P. aeruginosa genome. The study of the H1-T6SS has been facilitated by the fact it can be activated by the manipulation of the RetS/Gac/Rsm regulatory cascade by deletion of retS. However, the precise signals required for activation of this cascade, resulting in H1-T6SS activation, are unknown. This work investigates the role of subinhibitory concentrations of antibiotics in activating the system, and shows that kanamycin is able to induce production of core H1-T6SS components. This activation requires a functional Gac/Rsm cascade, but it is not known if this is due to direct signalling via the cascade, or due to a dominant effect of RsmA repression. The H2-T6SS is characterized in this work. We highlight key differences between the H2-T6SS cluster in PAO1 and PA14, including the presence of additional core T6SS components and putative secreted effectors. A strain is generated in which expression of the PA14 H2-T6SS cluster can be activated and tightly controlled by arabinose inducible promoters. The activity of the promoters is confirmed by the H2-T6SS dependent secretion of Hcp2 specifically upon arabinose induction. We further consider two putative H2-T6SS secreted substrates, VgrG14 and Rhs14. Production of these proteins is observed following arabinose induction, but their secretion is not detected. The Rhs14 protein is characterised, and its possible role as a H2-T6SS dependent effector is discussed. Finally, the H2-T6SS system in PA14 is shown to inhibit the internalisation of P. aeruginosa PA14, in contrast to the previously published observations of the H2-T6SS promoting internalisation of PAO1