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The CydDC transporter of Escherichia coli: investigating the impact of reductant export upon nitrosative stress, transcriptome/metabolome interplay, and host colonisation

Holyoake, Louise V. (2016) The CydDC transporter of Escherichia coli: investigating the impact of reductant export upon nitrosative stress, transcriptome/metabolome interplay, and host colonisation. Doctor of Philosophy (PhD) thesis, University of Kent,. (KAR id:55324)

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Abstract

CydDC of Escherichia coli is an ABC transporter that exports cysteine and glutathione from the cytoplasm to the periplasm to maintain redox homeostasis; its loss elicits a pleiotropic phenotype and causes the periplasm to become ‘over-oxidising’. In addition, the CydDC transporter is required for the assembly of cytochrome bd-I, a respiratory complex that provides tolerance to nitric oxide, a toxic radical produced by the host immune system in response to bacterial infection. The contribution of CydDC to nitric oxide tolerance and the pleiotropic phenotype of cydDC mutants implicates this exporter complex as a potential target for future therapies to combat E. coli infections. Indeed, given the rising incidence of multidrug-resistant bacterial infections, it is becoming increasingly important to develop novel strategies to combat infection. This thesis reports an investigation into the contribution of CydDC to NO tolerance, the relationship between CydDC expression and cytochrome bd-I assembly, adaptations resulting from loss of CydDC, and the requirement for CydDC for survival during infection.

The transcriptional response of cydDC mutants to exogenously added cysteine and glutathione was explored through microarray analysis in an attempt to gain a greater insight into the role of reduced thiol export to the periplasmic space. The addition of thiols affected genes involved in cell metabolism, respiration and led to the down-regulation of motility-related genes, providing insights into how the presence of CydDC substrates contribute to diverse cellular processes.

To investigate the contribution of CydDC to survival during infection, macrophage survival assays were performed along with an infection study using a mouse model of UTI (urinary tract infection). This work demonstrates that CydDC does not contribute to bacterial survival within NO-producing macrophage cells, and that loss of CydDC has no significant effect on the ability to colonise the mouse lower urinary tract. We therefore conclude that while CydDC is important for cytochrome bd-I assembly and NO tolerance in vitro, it may not be a suitable drug target to combat pathogenic strains of E. coli.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Shepherd, Mark
Uncontrolled keywords: CydDC Escherichia coli cysteine glutathione periplasm Nitric Oxide
Subjects: Q Science
Q Science > QR Microbiology
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Users 1 not found.
Date Deposited: 06 May 2016 17:00 UTC
Last Modified: 23 Jan 2020 04:11 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/55324 (The current URI for this page, for reference purposes)
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