Mechanisms of action of the Pseudomonas aeruginosa type III secreted toxins

Stirling, Fiona Ruth (2005) Mechanisms of action of the Pseudomonas aeruginosa type III secreted toxins. PhD thesis, University of Glasgow.

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Pseudomonas aeruginosa is a Gram-negative bacterium found ubiquitously in soil and water. It is an opportunistic pathogen that is capable of causing disease in susceptible humans, for example patients suffering from cystic fibrosis, burns or the immunocompromised. P. aeruginosa produces a large number of virulence factors that enable it to colonise and infect a wide range of tissue types. Among these virulence factors are the toxins secreted by the type III secretion (TTS) system. TTS systems are found exclusively in Gram-negative bacteria and they consist of a needle like structure that injects toxins from the bacteria directly into the cytoplasm of a eukaryotic target cell. P. aeruginosa encodes a TTS system that translocates four known toxins into eukaryotic cells; Exoenzyme (Exo) S, ExoT, ExoU and ExoY. ExoS and ExoT contain two catalytic domains, an N-terminal GTPase activating protein (GAP) domain and a C-terminal ADP-ribosyltransferase (ADPRT) domain. ExoU exhibits phospholipase activity and ExoY is an adenylate cyclase. All the TTS toxins from P. aeruginosa require a eukaryotic cofactor for activation. The ADPRT domains of ExoS and ExoT require a member of the 14-3-3 protein family, while the eukaryotic cofactors for ExoU and ExoY remain unknown. In this study we sought to further elucidate the mechanisms of action of the pseudomonal TTS toxins ExoS, ExoU and ExoY. Initially we used Saccharomyces cerevisiae as a model in which to study ExoS. We demonstrated that ExoS is so toxic to cerevisiae that the galactose-inducible GALl promoter system produced enough ExoS under glucose-repressing conditions to prevent transformation of an ExoS expressing construct. We therefore utilised a tetracycline-regulated activator-repressor dual system to provide tight control of ExoS expression and demonstrated that ExoS was indeed highly toxic to yeast. Both the GAP and ADPRT domains of ExoS were cytotoxic to S. cerevisiae but the ADPRT domain was responsible for the extreme potency of this toxin. We demonstrated that the ADPRT domain of ExoS disrupted the actin cytoskeleton by causing large aggregates of densely stained cortical actin patches, thick disorganised actin cables and a general loss in actin polarity. The actin disruption phenotype was similar to that of a yeast mutant that expresses mutant actin that is unable to disassociate and therefore forms very stable actin fibres. Thus, the ADPRT domain of ExoS may act by stabilising filamentous actin in S. cerevisiae. ExoS also caused an increase in the number of mating projections formed after treatment with the a-factor mating pheromone and inhibited normal bud formation after release from a- factor induced cell cycle arrest. Finally, using the S. cerevisiae system we showed that the ExoS ADPRT domain inhibits DNA synthesis following release from pheremone- induced growth arrest. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Microbiology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Evans, Prof. Tom
Date of Award: 2005
Depositing User: Enlighten Team
Unique ID: glathesis:2005-70957
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 08 May 2019 09:21
Last Modified: 25 Aug 2021 10:55
Thesis DOI: 10.5525/gla.thesis.70957
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