Causalities of war: The connection between type VI secretion system and microbiota

Cellular Microbiology published by John Wiley & Sons Ltd Microbiota niches have space and/or nutrient restrictions, which has led to the coevolution of cooperation, specialisation, and competition within the population. Different animal and environmental niches contain defined resident microbiota that tend to be stable over time and offer protection against undesired intruders. Yet fluxes can occur, which alter the composition of a bacterial population. In humans, the microbiota are now considered a key contributor to maintenance of health and homeostasis, and its alteration leads to dysbiosis. The bacterial type VI secretion system (T6SS) transports proteins into the environment, directly into host cells or can function as an antibacterial weapon by killing surrounding competitors. Upon contact with neighbouring cells, the T6SS fires, delivering a payload of effector proteins. In the absence of an immunity protein, this results in growth inhibition or death of prey leading to a competitive advantage for the attacker. It is becoming apparent that the T6SS has a role in modulating and shaping the microbiota at multiple levels, which is the focus of this review. Discussed here is the T6SS, its role in competition, key examples of its effect upon the microbiota, and future avenues of researc

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

Diversity and prevalence of type VI secretion system effectors in clinical Pseudomonas aeruginosa isolates

Pseudomonas aeruginosa is an opportunistic pathogen and a major driver of morbidity and mortality in people with Cystic Fibrosis (CF). The Type VI secretion system (T6SS) is a molecular nanomachine that translocates effectors across the bacterial membrane into target cells or the extracellular environment enabling intermicrobial interaction. P. aeruginosa encodes three T6SS clusters, the H1-, H2- and H3-T6SS, and numerous orphan islands. Genetic diversity of T6SS-associated effectors in P. aeruginosa has been noted in reference strains but has yet to be explored in clinical isolates. Here, we perform a comprehensive bioinformatic analysis of the pangenome and T6SS effector genes in 52 high-quality clinical P. aeruginosa genomes isolated from CF patients and housed in the Personalised Approach to P. aeruginosa strain repository. We confirm that the clinical CF isolate pangenome is open and principally made up of accessory and unique genes that may provide strain-specific advantages. We observed genetic variability in some effector/immunity encoding genes and show that several well-characterised vgrG and PAAR islands are absent from numerous isolates. Our analysis shows clear evidence of disruption to T6SS genomic loci through transposon, prophage, and mobile genetic element insertions. We identified an orphan vgrG island in P. aeruginosa strain PAK and five clinical isolates using in silico analysis which we denote vgrG7, predicting a gene within this cluster to encode a Tle2 lipase family effector. Close comparison of T6SS loci in clinical isolates compared to reference P. aeruginosa strain PAO1 revealed the presence of genes encoding eight new T6SS effectors with the following putative functions: cytidine deaminase, lipase, metallopeptidase, NADase, and pyocin. Finally, the prevalence of characterised and putative T6SS effectors were assessed in 532 publicly available P. aeruginosa genomes, which suggests the existence of accessory effectors. Our in silico study of the P. aeruginosa T6SS exposes a level of genetic diversity at T6SS genomic loci not seen to date within P. aeruginosa, particularly in CF isolates. As understanding the effector repertoire is key to identifying the targets of T6SSs and its efficacy, this comprehensive analysis provides a path for future experimental characterisation of these mediators of intermicrobial competition and host manipulation

Current landscape in the discovery of novel antibacterial agents

Background: Standard treatments against bacterial infections are becoming ineffective due to the rise of antibacterial resistance worldwide. Classical approaches to develop new antibacterial agents are not sufficient to fulfil the current pipeline, therefore new strategies are currently being devised in the field of antibacterial discovery. Objectives: The objective of this narrative review is to compile the most successful strategies for drug discovery within the antibacterial context that are currently being pursued. Sources: Peer-reviewed publications from the MEDLINE database with robust data addressing the discovery of new antibacterial agents in the current pipeline have been selected. Content: Several strategies to discover new antibacterials are described in this review: (i) derivatives of known antibacterial agents; the activity of a known antimicrobial agent can be improved through two strategies: (a) the modification of the original chemical structure of an antimicrobial agent to circumvent antibacterial resistance mechanisms and (b) the development of a compound that inhibits the mechanisms of resistance to an antibacterial agent; (ii) new antibacterial agents targeting new proteins; (iii) inhibitors of virulence factors; (iv) nanoparticles; (v) antimicrobial peptides and peptidomimetics; (vi) phage therapy and enzybiotics; and (vii) antisense oligonucleotides. Implications: This review intends to provide a positive message affirming that several different strategies to design new antibacterial agents are currently being developed, and we are therefore confident that in the near future some of the most promising approaches will come to fruition

Bacterial pathogenesis of plants: Future challenges from a microbial perspective

Plant infection is a complicated process. Upon encountering a plant, pathogenic microorganisms must first adapt to life on the epiphytic surface, and survive long enough to initiate an infection. Responsiveness to the environment is critical throughout infection, with intracellular and community-level signal transduction pathways integrating environmental signals and triggering appropriate responses in the bacterial population. Ultimately, phytopathogens must migrate from the epiphytic surface into the plant tissue using motility and chemotaxis pathways. This migration is coupled to overcoming the physical and chemical barriers to entry into the plant apoplast. Once inside the plant, bacteria use an array of secretion systems to release phytotoxins and protein effectors that fulfil diverse pathogenic functions (Fig. 1)(Phan Tran et al., 2011, Melotto & Kunkel, 2013)

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

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

Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate

Bacterial defences: mechanisms, evolution and antimicrobial resistance

Throughout their evolutionary history, bacteria have faced diverse threats from other microorganisms, including competing bacteria, bacteriophages and predators. In response to these threats, they have evolved sophisticated defence mechanisms that today also protect bacteria against antibiotics and other therapies. In this Review, we explore the protective strategies of bacteria, including the mechanisms, evolution and clinical implications of these ancient defences. We also review the countermeasures that attackers have evolved to overcome bacterial defences. We argue that understanding how bacteria defend themselves in nature is important for the development of new therapies and for minimizing resistance evolution