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

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. To gain a better understanding of how CydDC expression influences cytochrome bd-I assembly, cydDC cells were grown in the presence of exogenous cysteine and/or reduced glutathione. This work demonstrates for the first time that addition of cysteine and glutathione (i.e. both CydDC substrates) is necessary for cytochrome bd-I assembly in a cydDC strain. In vitro growth curves utilising a nitric oxide donor show that CydDC-mediated reductant export contributes to the tolerance of nitric oxide (NO) both via permitting cytochrome bd-I assembly and through a mechanism independent of this respiratory complex. This work is consistent with a model whereby NO-reactive thiols (i.e. cysteine and glutathione) exported to the periplasm can diminish the levels of NO that can enter the cytoplasm. 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

CydDC-mediated reductant export in Escherichia coli controls the transcriptional wiring of energy metabolism and combats nitrosative stress

The glutathione/cysteine exporter CydDC maintains redox balance in Escherichia coli. A cydD mutant strain was used to probe the influence of CydDC upon reduced thiol export, gene expression, metabolic perturbations, intracellular pH homeostasis, and tolerance to nitric oxide (NO). Loss of CydDC was found to decrease extracytoplasmic thiol levels, whereas overexpression diminished the cytoplasmic thiol content. Transcriptomic analysis revealed a dramatic up-regulation of protein chaperones, protein degradation (via phenylpropionate/phenylacetate catabolism), ?-oxidation of fatty acids, and genes involved in nitrate/nitrite reduction. 1H NMR metabolomics revealed elevated methionine and betaine and diminished acetate and NAD+ in cydD cells, which was consistent with the transcriptomics-based metabolic model. The growth rate and ?pH, however, were unaffected, although the cydD strain did exhibit sensitivity to the NO-releasing compound NOC-12. These observations are consistent with the hypothesis that the loss of CydDC-mediated reductant export promotes protein misfolding, adaptations to energy metabolism, and sensitivity to NO. The addition of both glutathione and cysteine to the medium was found to complement the loss of bd -type cytochrome synthesis in a cydD strain (a key component of the pleiotropic cydDC phenotype), providing the first direct evidence that CydDC substrates are able to restore the correct assembly of this respiratory oxidase. These data provide an insight into the metabolic flexibility of E. coli , highlight the importance of bacterial redox homeostasis during nitrosative stress, and report for the first time the ability of periplasmic low molecular weight thiols to restore haem incorporation into a cytochrome complex

The CydDC Family of Transporters and Their Roles in Oxidase Assembly and Homeostasis

The CydDC complex of Escherichia coli is a heterodimeric ATP-binding cassette type transporter (ABC transporter) that exports the thiol-containing redox-active molecules cysteine and glutathione. These reductants are thought to aid redox homeostasis of the periplasm, permitting correct disulphide folding of periplasmic and secreted proteins. Loss of CydDC results in the periplasm becoming more oxidising and abolishes the assembly of functional bd-type respiratory oxidases that couple the oxidation of ubiquinol to the reduction of oxygen to water. In addition, CydDC-mediated redox control is important for haem ligation during cytochrome c assembly. Given the diverse roles for CydDC in redox homeostasis, respiratory metabolism and the maturation of virulence factors, this ABC transporter is an intriguing system for researchers interested in both the physiology of redox perturbations and the role of low-molecular-weight thiols during infection