Targeting the Rho1 GTPase signalling pathway in Aspergillus fumigatus

Abstract

For individuals with compromised immune systems, Aspergillus fumigatus has emerged as one of the most common airborne fungal pathogens in recent decades. In immunosuppressed patients, inhalation of A. fumigatus spores can initiate life-threatening invasive aspergillosis (IA) with mortality rates up to 90%. The high mortality is largely due to a limited arsenal of antifungal drugs and emerging resistance and also the poor diagnosis. At present, the treatment for this fungal infection is dominated by three antifungal classes. Polyenes and azoles target the fungal cell membrane, and echinocandins target β-glucan synthesis in the fungal cell wall. However, they all have significant drawbacks in terms of toxicity, drug-drug interactions and/or efficacy.

The current efforts are to structurally and chemically validate new targets that are genetically proven as essential in A. fumigatus. One such protein AfRho1 is a Rho GTPase, plays essential roles in fungal physiology and cell wall organization. In this research, I combined X-ray crystallography and fragment-based small molecule screening by BLI (Biolayer interferometry) to investigate the ligandability of AfRho1. Using X-ray crystallography, the structure of AfRho1 was solved and a complex structure with one of the hit fragments was obtained. The complex structure showed that this fragment molecule bound to AfRho1 in a 2:1 molar ratio, and the fragment binding pocket is conserved with the human orthologue protein HsRhoA. Structure analysis shows that the pocket is located at the previously validated interaction interface between Rho GTPases and the upstream activation GEF (Guanine nucleotide exchange factor) proteins. Therefore, in this research I have identified a lead molecule that could be used to develop inhibitors against AfRho1 and HsRhoA by inhibiting the corresponding GEF proteins mediated nucleotide exchange.

Rom2, a Rho1 GTPase guanine nucleotide exchange factor (GEF), plays an essential role in the fungal cell wall integrity pathway and possesses a unique C-terminal CNH (Citron homology) domain. Using genetic and molecular approaches I characterised the functions of the Rom2 CNH domain in the pathogenic fungus Aspergillus fumigatus. Cell wall analysis including TEM and chemical analysis suggest that this domain is important for both β-glucan and chitin synthesis. GFP pull-down mass spectrometry and in vitro binding assays suggest that the CNH domain is a Rho1 GTPase binding protein. The protein structure of AfRom2 CNH domain was solved by X-ray crystallography. This revealed that the structure of AfRom2 CNH domain is a seven bladed WD40 protein that shares structural similarities to the GTPase binding protein β-transducin. From structure comparison of the AfRom2 CNH domain and AfRho1 to the heterotrimeric transducin complex, I discovered that the top surface of the CNH domain and an α-helix in the switch II region of AfRho1 may form the potential interaction surface. My work provides both genetic evidence and the molecular basis that the A. fumigatus Rom2 CNH domain regulates cell wall synthesis, therefore validate it as a potential antifungal target.

Date of Award	2017
Original language	English
Awarding Institution	University of Dundee
Sponsors	China Scholarship Council
Supervisor	Daan van Aalten (Supervisor)