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

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

Proteomic Analysis of Resistance of Gram-Negative Bacteria to Chlorhexidine and Impacts on Susceptibility to Colistin, Antimicrobial Peptides, and Ceragenins

Use of chlorhexidine in clinical settings has led to concerns that repeated exposure of bacteria to sub-lethal doses of chlorhexidine might result in chlorhexidine resistance and cross resistance with other cationic antimicrobials including colistin, endogenous antimicrobial peptides (AMPs) and their mimics, ceragenins. We have previously shown that colistin-resistant Gram-negative bacteria remain susceptible to AMPs and ceragenins. Here, we investigated the potential for cross resistance between chlorhexidine, colistin, AMPs and ceragenins by serial exposure of standard strains of Gram-negative bacteria to chlorhexidine to generate resistant populations of organisms. Furthermore, we performed a proteomics study on the chlorhexidine-resistant strains and compared them to the wild-type strains to find the pathways by which bacteria develop resistance to chlorhexidine. Serial exposure of Gram-negative bacteria to chlorhexidine resulted in four- to eight-fold increases in minimum inhibitory concentrations (MICs). Chlorhexidine-resistant organisms showed decreased susceptibility to colistin (8- to 32-fold increases in MICs) despite not being exposed to colistin. In contrast, chlorhexidine-resistant organisms had the same MICs as the original strains when tested with representative AMPs (LL-37 and magainin I) and ceragenins (CSA-44 and CSA-131). These results imply that there may be a connection between the emergence of highly colistin-resistant Gram-negative pathogens and the prevalence of chlorhexidine usage. Yet, use of chlorhexidine may not impact innate immune defenses (e.g., AMPs) and their mimics (e.g., ceragenins). Here, we also show that chlorhexidine resistance is associated with upregulation of proteins involved in the assembly of LPS for outer membrane biogenesis and virulence factors in Pseudomonas aeruginosa. Additionally, resistance to chlorhexidine resulted in elevated expression levels of proteins associated with chaperones, efflux pumps, flagella and cell metabolism. This study provides a comprehensive overview of the evolutionary proteomic changes in P. aeruginosa following exposure to chlorhexidine and colistin. These results have important clinical implications considering the continuous application of chlorhexidine in hospitals that could influence the emergence of colistin-resistant strains

Investigation into the changing colonisation of skin bacteria during isotretinoin treatment for acne vulgaris

Poster presentatio

The Pseudomonas aeruginosa T6SS Delivers a Periplasmic Toxin that Disrupts Bacterial Cell Morphology.

The type VI secretion system (T6SS) is crucial in interbacterial competition and is a virulence determinant of many Gram-negative bacteria. Several T6SS effectors are covalently fused to secreted T6SS structural components such as the VgrG spike for delivery into target cells. In Pseudomonas aeruginosa, the VgrG2b effector was previously proposed to mediate bacterial internalization into eukaryotic cells. In this work, we find that the VgrG2b C-terminal domain (VgrG2bC-ter) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein. We resolve the structure of VgrG2bC-ter and confirm it is a member of the zinc-metallopeptidase family of enzymes. We show that this effector causes membrane blebbing at midcell, which suggests a distinct type of T6SS-mediated growth inhibition through interference with cell division, mimicking the impact of β-lactam antibiotics. Our study introduces a further effector family to the T6SS arsenal and demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells

Environmental Implications of Francisella Tularensis Biofilms

Francisella tularensis survives in one of the widest environmental ranges of any pathogen. Numerous mammals and arthropod vectors are infected by this highly virulent organism. How this zoonotic pathogen persists outside of its many hosts remains unexplored. We aimed to examine how F. tularensis interacts with environmental surfaces, and hypothesized that biofilm formation may enable survival of this organism in nature. By understanding the role these surface-attached bacterial communities play in F. tularensis ecology, we hope to gain insight into the mechanisms of environmental persistence and transmission of this pathogen. We identify chitin as a potential non-host niche for F. tularensis in nature using genetic, microscopic, and biochemical techniques. This abundant polysaccharide supported F. tularensis biofilm formation in the absence of an exogenous carbon source. This interaction was dependent on putative chitinase enzymes which hydrolyze the glycosidic bonds that connect GlcNAc monomers. Using a genetic screen, we identified adherence factors, including FTN_0308 and FTN0714 that promote attachment to chitin and colonization of chitin surfaces. We propose that biofilm formation on chitin surfaces in nature enables nutrient scavenging in oligotrophic environments allowing this pathogen to replicate and seed disease transmission. We found that the effect of nutrient limitation on F. tularensis biofilm formation extended beyond chitin utilization. Genetic studies indicated that nutrient starvation triggers a biofilm stress response. We identified static growth and nutrient deprivation as cues for enhanced biofilm formation. Microarray expression studies identified genes highiy expressed under these conditions, including F. tularensis biofilm determinants. Expression of nutrient transporters further indicated that biofilm formation promotes environmental persistence. We finally examined statically grown F. tularensis microscopically to determine if altered morphology explained the enhanced biofilm phenotype of these cultures. We discovered a novel F. tularensis appendage conserved between subspecies and structurally homologous to the Caulobacter crescentus stalk. These structures were observed in association with surfaces during both biofilm formation and during intracellular infection. A genetic screen for mutants in stalk formation revealed that stalk biosynthetic components are essential. We predict this structure aids in environmental persistence by facilitating surface attachment and nutrient uptake. Through this collective work we define evidence that surface association via biofilm formation promotes survival during nutrient limitation

07. Environmental Implications of Francisella Tularensis Biofilms

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