Sensory perception in model and pathogenic fungi: Engineering misappropriation of response

Abstract

The research described characterises pH signalling in the human pathogen Aspergillus fumigatus. Normal functioning of this regulatory system in Aspergillus species requires the integrity of seven genes, including the DNA binding transcription factor PacC, which responds to an alkaline pH signal transmitted by PalA, B, C, F, H and I. Two protein complexes are proposed to sense and transmit the pH signal, the first of which is plasma membrane-localised and composed of two putative pH signal receptors, PalH and PalI, plus a cytoplasmic non-metazoan member of the arrestin family, PalF. Using A. fumigatus transcriptional profiling, Saccharomyces cerevisiae phenotypic screening and the split-ubiquitin MYTH technology, A. fumigatus functions, likely to moderate, or be moderated by the PacC transcription factor, were sought. Two independent A. fumigatus ΔpacC strains have been phenotypically characterised and assessed for virulence in a neutropenic murine model of pulmonary aspergillosis. Of the two, one mutant was further characterised using transcriptional profiling in order to elucidate functions under PacC control during murine infection. The results of this analysis underline the physiological importance of alkaline adaptation during initiation of infection, especially with respect to impacts upon cellular homeostasis, transport and nutrient availability. A S. cerevisiae membrane two-hybrid system has been developed to investigate A. fumigatus plasma membrane protein-protein interactions. Applying the technology, the network of interactions between the components of the A. fumigatus plasma membrane pH signalling complex was explored, and the interaction between the pH-sensing PalH protein and the cytoplasmic arrestin PalF and likely the homodimerisation of PalH were identified. A screen for novel interactors of PalH was also performed, providing 5 candidates for further validation. The S. cerevisiae Δrim101 null mutant and another 80 cation-sensitive null mutants were analysed. An epistacy screen to address the impact of activated Rim101p upon cation sensitivity was performed supporting the identification of putative novel Rim101p/PacC regulators. In parallel the activity of a synthetic Rim101p-regulatable promoter was examined identifying 10 mutants suffering aberrant Rim101p processing and/or transcriptional activity. Finally two A. fumigatus strains have been constructed to functionally express the firefly luciferase gene. Preliminary tests indicate this as a powerful resource to follow murine infection in real time with bioluminescence assays.