AbstractCampylobacter jejuni is a leading cause of food poisoning and according to the World Health Organisation accounts for majority of the 4.5 billion cases of global food poisoning each year. Genome sequencing by Parkhill et al. (2000) identified a gene, cj1411c, which is thought to encode a lone cytochrome P450, CYP172A1. In this thesis the role of CYP172A1 was studied using in vivo and in vitro techniques. The genomic location of cj1411c is adjacent to the capsular biosynthetic genes. The capsular and P450 genes are conserved in some species of Campylobacter and Helicobacter, as well as in Comamonas testosteroni. Importantly, this work has demonstrated that the P450 gene is expressed in two well characterised laboratory C. jejuni strains, 11168H and 81-176. Protein production was disrupted using insertional knockout mutagenesis, which allowed for investigations into the role of the enzyme in the host. Alterations to the observed autoagglutination rate and growth characteristics indicated that CYP172A1 has a role in modifying the bacterial surface. The insertional knockout mutant also resulted in cells which were more susceptible to detergent-like compounds (e.g. polymyxin B and sodium deoxycholate). In a previous report, it was suggested that the loss of the P450 function resulted in bacteria which were "shorter and fatter", compared to wild type cells, but this thesis could find no evidence of such a phenomenon. CYP172A1 was successfully purified using recombinant expression in E. coli to enable biochemical and biophysical characterisation in vitro. CYP172A1 contains a typical P450 cysteine thiolate coordination to the heme iron, and exists in a low spin ferric heme state under neutral buffer conditions. The P450 was found to self aggregate, and despite rigorous investigations the cause of this aggregation was not fully established. Despite this issue, CYP172A1 was shown to bind to a wide range of P450 inhibitor-type compounds, with econazole displaying the tightest binding affinity (Kd = 100 nM). Identification of substrate-like compounds was achieved using high throughput compound screening, and a number of organic compounds were identified and shown to bind CYP172A1, inducing heme iron absorbance changes typical of either P450 inhibitors or substrates. Optical titrations for these molecules indicated that their CYP172A1 Kd values were in the low micromolar range. The catalytic capability of CYP172A1 was successfully demonstrated by providing the P450 with non native redox partners to oxidise one of such substrate-like compound (213071), resulting in the sulfoxidation of this compound.
|Date of Award||31 Dec 2013|
|Supervisor||Andrew Munro (Supervisor) & James Linton (Supervisor)|
- Cytochrome P450
- Campylobacter jejuni