From Bug to Drug: Identification of novel inhibitors of pH signalling in the fungal pathogen Aspergillus fumigatus.

Abstract

In fungal pathogens of insects, plants, and animals, pH adaptation is a critical virulence determinant. In Aspergillus fumigatus mutants lacking the pH-sensing 7TMD receptor PalH, or pH-responsive transcription factor PacC, demonstrate tissue non-invasive phenotypes and significantly attenuated virulence in murine models of aspergillosis. Inhibition of pH signalling therefore represents an attractive antifungal strategy. To promote efficient development of urgently required novel antifungals, expansion of a genetic toolbox that enables mechanistic understanding of virulence-essential processes, and allows their perturbation, is required. To this end, this study developed a fluorescent reporter, ena1P_YFP, of A. fumigatus pH signalling functionality constructed in an isogenic set of pH signalling and non-signalling (WT, pacC -/- and palH -/-) in the A. fumigatus ATCC46645 genetic background, with which to measure functionality of PacC mediated gene expression and which was subsequently instrumental in establishing drug mode of action. Using pH signalling as a prototype, a universally applicable methodology was sought with which to capture A. fumigatus protein-protein interactions in high throughput via bimolecular fluorescence complementation. The pH receptor- and arrestin-encoding genes, palH and palF respectively, were cloned as translational fusions to the NH2- and COOH- termini of a split yellow fluorescent protein (YFP) and co-transformed, on stably maintained episomal plasmids, into corresponding null mutants of A. fumigatus. Functional restoration of pH non-signalling phenotypes was achievable under alkaline growth conditions but despite efficient co-maintenance of genetically distinct episomal species, functional reconstitution of YFP was not possible. Via CRISPR-CAS9 mediated genomic editing the pH dependent localisation of PacC has been further characterised, and nuclear localisation of the transcription factor captured following alkaline shift. A GFP_PacC fusion protein expressed via a gpdA mini promoter, revealed a plasma membrane-proximal localisation of PacC under acidic environmental conditions, thereby substantiating previous hypotheses on the subcellular site of signalling proteolysis. In A. nidulans, under alkaline conditions, PacC directly represses the expression of gabA, a gene which encodes a gamma-aminobutyric acid (GABA) permease. Consequently, loss of function pacC mutations promote GABA uptake, via gabA de-repression, providing a convenient growth reporter of PacC signalling functionality. Exploiting this regulatory paradigm a novel screen for inhibitors of Aspergillus pH signalling was devised and optimised in 384 well format. Having confirmed functional complementarity of A. fumigatus and A. nidulans PalH this was then exploited to transpose a screen for inhibitors of a pathogenic signalling mechanism into a genetically and practically tractable model Aspergillus species to facilitate screening and mode of action studies. A small molecule fragment library was screened and hits identified, that evoked a pH non-signalling phenotype. Through industrial collaboration, and an iterative hit identification process, a novel structure-activity relationship has been identified that conform to the mode of action expected of hits which perturb pH signalling. To conclude, innovative methods are required to find novel inhibitors of pathogenically important mechanisms. In genetically intractable fungi, this requires significant effort and is thus not amenable to high throughput approaches. This study aimed to develop a genetic toolbox by which both characterization of pH signalling functionality and novel pH signalling-targeted therapeutic agents is possible. Consequently, we have developed a means to reverse engineer the pH signalling pathway of A. nidulans when the native pH sensing palH gene has been replaced with that of A. fumigatus, enabling identification of molecules with the ability to regulate PacC mediat

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Nick Read (Supervisor) & Elaine Bignell (Supervisor)