The disulfide oxidative pathway in Chlamydia trachomatis

Abstract

The disulfide oxidative pathway in bacteria is responsible for disulfide bond formation in secreted proteins including many virulence factors. The archetypal Escherichia coli DsbA and DsbB enzymes form a redox relay that catalyses disulfide bond formation and constitutes the oxidative folding pathway. DsbA orthologs from a broad range of bacteria have been characterised and while the enzymes have similar structural and biochemical characteristics they vary in redox chemistry and surface properties. An overview of the DsbA and DsbB enzymes from different bacteria is provided in Chapter 1. Chapter 1 also provides an overview of antibiotics with well-known and novel targets, including bacterial oxidative folding.Chlamydia trachomatis is an obligate intracellular pathogen responsible for blinding trachoma and the sexually transmitted infection chlamydia that can lead to infertility and ectopic pregnancy. C. trachomatis has a biphasic developmental cycle that is dependent on coordinated disulfide bond formation and reduction for differentiation between the two distinct cell types: reticular body (RB) and elementary body (EB) (reviewed in chapter 1).Chapter 2 presents a biochemical and structural characterisation of a truncated soluble form of DsbA from C. trachomatis (CtDsbA), which was published in PLOS ONE. CtDsbA is the first structurally characterised DsbA having two small, uncharged amino acids separating the two active site cysteines (Cys-Ser-Ala-Cys). Characterisation of CtDsbA shows that is has oxidase activity and is structurally similar to other DsbAs. However, CtDsbA is distinct in that it is the weakest oxidising DsbA so far described. This is consistent with the analysis of the 2.7 A resolution crystal structure showing a lack of factors stabilising the nucleophilic thiolate anion of Cys38. CtDsbA is also distinguished from most other DsbAs by having a disulfide bond, linking helix 2 and helix 5, that has only been reported in three other structurally characterised DsbAs, including Wolbachia pipientis DsbA1 (WpDsbA1).Chapter 3 describes my studies of the interaction between CtDsbA and CtDsbB. In the oxidative pathway of E. coli, DsbA (EcDsbA) is kept in its active, oxidised state by the integral membrane protein DsbB (EcDsbB) in a mechanism dependent on a quinone co-factor. A BLAST search identified a protein (CtDsbB) in the genome of C. trachomatis with 21% sequence identity to EcDsbB and a predicted secondary structure topology equivalent to EcDsbB. Chapter 3 presents the characterisation of the interaction between CtDsbA and CtDsbB showing that crude membranes containing heterogeneously expressed CtDsbB are able to oxidize CtDsbA. However, purified, detergent solubilised CtDsbB is not able to oxidise CtDsbA in the presence of ubiquinone-1 suggesting that purified CtDsbB is inactive in detergent micelles or that ubiquinone-1 is not a suitable co-factor for CtDsbB. In contrast to what was found for WpDsbA1, the non-catalytic disulfide of CtDsbA does not regulate interaction with CtDsbB. Interestingly, mutating the cysteines forming the non-catalytic disulfide to serines has minimal effect on CtDsbA thermal stability, and only marginally decreases the difference in melting temperature between reduced and oxidised forms of the enzyme.In Chapter 4, I report on my attempts to generate a dsbA loss-of-function mutant. As disulfide cross-linking of the outer membrane plays an important role in C. trachomatis development and infection, CtDsbA might play a specialised role to regulate the redox state of the cysteines that form cross-links in the outer membrane. This hypothesis is supported by the observation I made that CtDsbA expression correlates with RB to EB differentiation (Chapter 3). To pursue this further, I attempted to obtain a loss-of-function dsbA mutant by chemical mutagenesis with ethyl methanesulfonate (EMS). In a forward genetics approach, using sensitivity to dithiothreitol (DTT) as a phenotypic read out, approximately 2000 subpopulations of EMS treated C. trachomatis were screened. 14 subpopulations exhibited increased sensitivity towards DTT. Despite a hypothesis that a loss-of-function dsbA mutant would exhibit increased sensitivity to DTT, none of the 14 subpopulations had a mutation in the dsbA gene. In a reverse genetics approach approximately 14400 subpopulations were screened using Targeting Induced Local Lesions in Genomes (TILLING), but again no dsbA mutation was identified.CtDsbA is the first DsbA to be characterised that has a catalytic dipeptide containing two small, uncharged amino acid residues. Characterisation revealed that CtDsbA is weakly oxidising with a mildly destabilising active site disulfide. Hence, characterisation of CtDsbA provides new insight into the diversity amongst DsbA proteins and supports the ongoing effort to develop inhibitors of DsbA enzymes. By comparison to E. coli, CtDsbB can form a redox-relay with CtDsbA. The forming of a functional redox relay was confirmed by the finding that crude membranes expressing CtDsbB facilitates CtDsbA activity. Despite screening more than 16000 subpopulations of EMS treated C. trachomatis, no mutant was identified in the dsbA gene. Further experiments will be required to unpick the biological role of CtDsbA, and I discuss what these experiments could entail in Chapter 5.