Structure-function analysis of a pH-responsive molecular switch required for fungal virulence in Aspergillus fumigatus

Abstract

Ambient pH signalling is a crucial determinant of Aspergillus fitness in alkaline environments such as the mammalian host. Ambient pH signalling is governed by PacC, activation of which leads to the synthesis of extracellularly-acting enzymes and permeases. The functionality of the signalling mechanism requires six pal gene products, including a GPCR-like protein PalH, an arrestin-like protein PalF and an integral membrane tether protein PalI. In ascomycetous yeasts the functionality of PalH homologues is dependent upon interaction with a second seven transmembrane domain (7TMD) protein Dfg16 that is absent in Aspergillus spp. Based upon prior evidence of PalH homodimerisation this study sought to detect the presence (Chapters 3, 4), and functional significance of PalH oligomers (Chapters 5, 6, 7). To determine the expression, stability and molecular weight of membrane-based pH signalling components, and complexes thereof, various membrane protein extraction and gel electrophoresis strategies were tested and optimised (Chapter 3). The stability and indicative molecular weight of HA3-tagged A. fumigatus PalH under denaturing and non-denaturing electrophoresis were tested and a stably expressed complex of a molecular weight of 172.5 kDa was detected under non-denaturing gel electrophoresis. To further substantiate the occurrence of PalH oligomers in Aspergillus sp. the parasexual cycle of Aspergillus nidulans was exploited to co-express differentially-tagged A. nidulans PalH receptors (Chapter 4). Co-expression of HA3- and Myc3-tagged PalH proteins, co-immunoprecipitation (co-IP) and Westen blotting (WB) revealed that PalH-HA3 can be co-immunoprecipitated with PalH-Myc3 and PalH-Myc3 can be co-immunoprecipitated with PalH-HA3. Hence, the oligomerisation between differently epitope-tagged PalH proteins of A. nidulans was clearly revealed for the first time in this work. To determine whether oligomerisation of A. fumigatus PalH is required for functionality, six truncated Tet_OFF-palH-HA3 alleles (TA to TF) were constructed and expressed in a palH null isolate to test the capacity of the truncated palH alleles to rescue the alkaline sensitivity of the recipient strain (Chapter 5). The result showed that the integrity of all transmembrane domains (TMDs) of PalH and the PalH N-terminus are indispensable for PalH functionality. However, the first 73 amino acids of the N-terminus region of PalH were dispensable for signalling. Next, all Tet_OFF::palH-HA3 truncation alleles were expressed in a WT background. Phenotypic analyses revealed a dominant negative phenotype of a TE-PalH-HA3 protein when co-expressed with PalH native protein. The same mutant allele, expressed in a palH null background yielded a complete loss of function phenotype. Supported by WB data, this finding suggests that the N-terminally truncated PalH receptor interferes with the function of co-expressed wild-type PalH receptors via the formation of dysfunctional hetero- and/or homo-oligomers. This study also aimed to determine whether the functionality of Aspergillus PalH proteins occurs as part of a pH sensing complex via homo- and/or hetero-oligomer formation involving PalF and/or PalI, and the requirement for such oligomerisation in ambient pH signalling (Chapters 6, 7). To explore the dependency of A. fumigatus PalH upon PalF and PalI, the expression of Tet_OFF-palH-HA3 in PalF and PalI null backgrounds was explored. PalH-HA3 was stably expressed in the absence of PalF but not in the absence of PalI. Additionally, for the first time, an A. fumigatus PalI null mutant was constructed and shown to exhibit a complete loss of pH signalling identifying a new target for drug discovery studies (Chapter 6). To determine the interdependency of PalH, PalF and PalI upon stable expression of oligomer components, the C-terminus of each protein was fused to a HA3 epitope, and the fusion proteins were expressed under the control of a Tet_OFF doxycycline responsive promoter. The c

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Michael Bromley (Supervisor) & Elaine Bignell (Supervisor)