Structural and functional studies of proteins expressed from the gly1 locus of pathogenic Neisseria

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.