Towards the development of novel Aspergillus fumigatus targeted antifungals with an in-depth analysis of Sfp-PPTase, PptA

Abstract

Humans are continuously confronted by the threat of fungal infection. Estimates put the number of individuals infected by superficial fungal disease at about 1.7 billion. Additionally there are a significant proportion of invasive infections which are difficult to treat and lead to an estimated 1.5 million deaths each year. Aspergillus fumigatus is an opportunistic, fungal pathogen which can lead to a variety of disease manifestations, generally termed aspergillosis and account for more than 200,000 life threatening infections annually with mortality rates of up to 95%. The occurrence of fungal disease has increased significantly due to the expansion of the immune deficient population, particularly those who receive immunosuppressive therapies. This combined with the problems surrounding current therapeutic options for fungal disease such as a rise in the incidence of antifungal resistance, adverse side effects in patients and drug-drug interactions has led to an urgent need to discover and develop an innovative class of antifungal agents. This thesis will address this requirement by validating a potential drug target, PptA, for combating A. fumigatus infection and explore methods to identify novel antifungal target identification. PptA is a Sfp-type 4'-Phosphopantetheinyl transferase required for transfer and covalent tethering of 4'-phosphopantetheine from coenzyme A to a conserved serine residue within a peptidyl carrier domain of a protein substrate. This project outlines the many critical roles PptA plays within A. fumigatus, such as; involvement in the production of many virulence factors as well as the biosynthesis of the essential amino acid, lysine. Furthermore, it is demonstrated that PptA is vital for secondary metabolite production in A. fumigatus, growth in iron limiting conditions and virulence. Finally, the design of a high-throughput screening assay capable of identifying inhibitors of PptA enzymatic activity further demonstrate the suitability of this target. It is the combined effect of all these characteristics that makes PptA such a suitable candidate for an antifungal target.Additionally two validation methods are included which can be used to identify novel drug targets. The first method utilises chemically induced haploinsufficiency profiling, a technique which has been proven successful for drug target identification and determining mode of action of drugs in S. cerevisiae and C. albicans. The project has shown this technology can be successfully used in A. fumigatus. Furthermore, a new technique for in vitro parallel fitness screening using next generation sequencing was validated with a library of phosphatase deletion mutants. Bioinformatic analysis against strains exhibiting severe fitness defects allowed the identification of three phosphatases that have the potential of becoming successful antifungal drug targets.

Date of Award	1 Aug 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Michael Bromley (Supervisor) & Paul Bowyer (Supervisor)