Characterisation of the transcriptional regulator CysB in Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted infection gonorrhoea. Rapid emergence of antibiotic resistant strains of N. gonorrhoeae has given rise to multi-drug resistant gonorrhoea. Combined with increasing incidence and the most recent resistance event in 2018, there is an urgent need for new antimicrobial treatments for gonorrhoeal infection. N. gonorrhoeae has a limited number of transcriptional regulators, one of which, CysB, is essential and regulates genes required for de novo cysteine biosynthesis. The cysteine biosynthetic pathway is a promising target for the development of novel antimicrobials. Cysteine plays an important role in protein folding and function, and is the precursor for many thiol-containing molecules within N. gonorrhoeae including effective oxidative stress defences such as glutathione. N. gonorrhoeae displays unique differences in assimilation of sulfur and cysteine biosynthesis, yet little is known about how this altered pathway is regulated in N. gonorrhoeae. The cysteine regulon consists of the genes required for sulfur transport, assimilation and cysteine biosynthesis. The transcriptional regulator CysB regulates this regulon in other organisms and is essential in N. gonorrhoeae. Understanding how N. gonorrhoeae regulates gene expression will improve our understanding of the pathogenicity of N. gonorrhoeae. In order to understand how CysB mediates gene expression, we need to characterise DNA-binding activity and elucidate the three-dimensional structure. In this thesis, we determine the DNA-binding activity of CysB, its three-dimensional structure and investigate conformational changes of CysB. Our results indicate that CysB regulates genes in the altered cysteine regulon, but that CysB does not respond to the inducer N-acetylserine. The three-dimensional structure of CysB is the first reported full-length CysB structure and was solved to 2.73 Å. Analysis of this structure, coupled with small angle scattering data suggest that CysB from N. gonorrhoeae lacks the ability to undergo the hypothesised large scale conformational changes upon inducer binding. This indicates that CysB may have an alternate mechanism in N. gonorrhoeae. Collectively, the data presented in this thesis represents key advances in our understanding of how N. gonorrhoeae regulates gene expression by characterisation of the transcriptional regulator CysB. Data presented here is the basis for future work to identify other targets within N. gonorrhoeae that may be investigated for the development of new antimicrobials for treatment of extensively drug resistant gonorrhoea.