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Neisseria meningitidis and Neisseria gonorrhoeae are closely related human-specific pathogens that colonise mucosal surfaces. N. meningitidis is the most common cause of bacterial meningitis and septicaemia in the UK, whilst N. gonorrhoeae causes sexually transmitted infections. In 2017 the latter was classified by the World Health Organization as a high priority organism for research and development of new treatments, as antibiotic resistance is rapidly spreading and several extensively drug resistant strains exist. The work described here focuses on the gly1 locus (gonolysin 1), which consists of two open reading frames (ORFs 1 and 2) first identified in N. gonorrhoeae and highly conserved in other pathogenic Neisseria. These genes are hypothesized to play a role in the battle between host and pathogen for the essential nutrient iron, an important factor in pathogenicity and the outcome of infection. Previous studies have shown that Gly1ORF1 binds haemin and may be important for N. meningitidis survival using haemin or haemoglobin as an iron source. The structure of the wild type N. meningitidis Gly1ORF1 protein has been solved in this study. Further characterisation of haemin binding function has been achieved through multiple biophysical techniques. Analysis of knock-out mutants created in this study show that unlike in N. meningitidis, Gly1ORF1 is not essential for utilisation of haemin or haemoglobin as iron sources in N. gonorrhoeae. Preliminary investigations to identify alternative functions have been carried out and provide scope for future work to identify the role of this conserved protein. The first reported study of gly1ORF2, which shares partial homology with the E. coli haem biosynthesis enzyme Uroporphyrinogen III synthase (UroS), is also presented here. Genetic complementation in E. coli has confirmed that it is the neisserial hemD homologue that encodes UroS. This is supported by the observation that N. gonorrhoeae null mutants require an exogenous haem source for survival. The protein has been purified and techniques including circular dichroism, bio-layer interferometry and bioinformatics have been employed to study its structure and interactions. Overall the results of this study add to our understanding of haem/iron metabolism in Neisseria and provide scope for further study of this area that may contribute to development of new treatments or prophylactics.

Kinetoplastid parasites pose a huge global health burden on both humans and livestock. Current treatments are toxic and difficult to administer. With the emergence of drug-resistant organisms, it is essential to identify new therapeutic targets. African trypanosomiasis, or sleeping sickness, is caused by Trypanosoma brucei. This thesis focuses on the characterisation and validation of a potential new drug target in this kinetoplastid parasite. Flap endonucleases are essential enzymes involved in DNA replication and repair. Synthetic constructs, encoding wild type and mutated FEN genes, were produced and expressed in Escherichia coli. Purification procedures and quantitative enzyme activity assays were developed. The catalytic and binding activity of the D183K variant were determined and compared to the wild type enzyme. The former was shown to be catalytically inert, yet able to bind a DNA substrate with similar affinity to the wild type enzyme (Kd = 2.22 and 1.37 μM respectively). The D183K variant was crystallised collaboratively and its structure determined by colleagues in the Department of Molecular Biology & Biotechnology. The wild type and D183K variant genes were over-expressed in T. brucei bloodstream form cells, using a tetracycline-inducible system. Expression of the catalytically inert protein had a severely detrimental effect on cell growth, and morphological changes were observed 72 hours post-induction. Knock-down assays were attempted using siRNA techniques, but results were inconclusive. The recombinant wild type enzyme was targeted with known FEN inhibitors. Myricetin, a commercially available flavonoid, inhibited T. brucei flap endonuclease (IC50 = 14.81 μM). T. brucei bloodstream form cells were killed upon exposure to myricetin (LD50 = 23.63 μM). A high-throughput thermal shift assay was developed to aid fragment-based drug design. This assay can be used to detect binding by myricetin, a known TbFEN inhibitor. Use of this assay will open up further avenues of exploration for targeting the T. brucei flap endonuclease.