Abstract
Energy generation is fundamental to all life on this planet, and oxidative phosphorylation via the electron transport chain is a universal process in all eukaryotes and prokaryotes. Due to its essential role in many cellular processes, the electron transport chain has become the target of antimicrobials for the control of both obligate human pathogens and pathogens affecting our precious crops. The protein complexes comprising the electron transport chain can differ between organisms, an advantageous feature, which can allow for the species-specific development of inhibitors targeting oxidative phosphorylation. This thesis examines the electron transport chains of Phytophthora agathidicida and Neisseria gonorrhoeae. Both pathogens urgently require new anti-infectives for the treatment of their respective diseases. Kauri dieback disease and gonorrhoea have become increasingly larger problems in Aotearoa, New Zealand. The impact and effects of these organisms also disproportionally affect Māori, our indigenous population.
P. agathidicida is the causative agent of kauri dieback, a disease which will likely cause the extinction of New Zealand’s native kauri trees if no curative agent is found. Currently phosphite injections are the only treatment option for infected trees, however the effect of these injections simply delays the progression of the fatal disease. Fungicides targeting complexes of the electron transport chain have been successfully used both in New Zealand and worldwide to protect and treat crops from similar Phytophthora diseases, however, none are used against kauri dieback disease. Here we characterise the respiratory chain of P. agathidicida and further demonstrate the inhibition of cytochrome bc1 and the F1Fo-ATP synthase by classical respiratory inhibitors at two different lifecycle stages of P. agathidicida. In addition, the compounds with potent activity against cytochrome bc1 were cyazofamid and fenamidone, two fungicides registered for use in New Zealand, further expanding their spectrum of activity to P. agathidicida.
N. gonorrhoeae causes the sexually transmitted infection gonorrhoea, which affects 78 million people a year. Since antibiotics were first prescribed for its treatment resistance has quickly followed. The Center for Disease Control and Prevention now has only one recommended treatment, and resistance has now started to emerge against this last line of defence. We took a similar approach as we did for P. agathidicida, determining the enzymes within its electron transport chain and measured the effectiveness of respiratory inhibitors against each complex. The cytochrome bc1 complex of N. gonorrhoeae was also effectively inhibited by cyazofamid and fenamidone. Reported inhibitor of the bc1 complex of Mycobacterium tuberculosis, TB47, was ineffective against the bc1 complex of N. gonorrhoeae, and cyazofamid and fenamidone were without effect on the mycobacterial bc1 complex. The specificity of these two cyazofamid and fenamidone, traditionally used as anti-oomycetes, was further analysed through in silico analysis. Binding of these inhibitors to a homology model of the Neisseria bc1 structure, was compared to the Mycobacterium tuberculosis structure. Key amino acid resides within the quinone- and quinol-binding pockets of bc1 likely prevented the effective inhibition of the M. tuberculosis cytochrome bc1 by these compounds and vice versa for TB47.
Overall, this thesis presents the respiratory chain complex cytochrome bc1 of P. agathidicida and N. gonorrhoeae as a potential new target for the development of anti-infectives.