In fungi, efficient adaption to extracellular pH combines cellular homeostasis and regulated genetic control, the latter exerted via a family of pH-responsive transcription factors PacC/Rim101. Aspergillus species monitor extracellular pH via 7 transmembrane domain (7-TMD) integral membrane proteins called PalH. These putative pH sensors activate PacC-mediated signaling via interaction with a cognate arrestin PalF, potentially acting as pH-dependent molecular switches required for pathogenicity. The aim of this study was to decipher the role of A. fumigatus PalH in PacC-mediated signaling and during mammalian infection.
ΔpalH mutants were tested for (i) PacC processing at different pHs (pH 3.5, 5.0 and 8.0) using electrophoretic mobility shift assay (EMSA), (ii) sensitivity to alkaline pH, salt stress and cell wall damaging agents, (iii) damage to epithelial A549 monolayers and (iv) virulence in a leukopenic murine model of pulmonary aspergillosis. To probe the mechanistic basis of PalH-mediated signalling, we utilised a split-ubiquitin Membrane Yeast Two-Hybrid (MYTH) assay to assess protein-protein interactions amongst candidate A. fumigatus signalling proteins of this pathway. To look for novel, membrane-proximal, regulators of PacC, a S. cerevisiae library of recombinant A. fumigatus preys was constructed and screened against a full length PalH bait. A. fumigatus null mutants of putative PalH interactors were constructed and subjected to phenotypic analysis at acidic and alkaline pH.
EMSA analysis indicated an absolute requirement for PalH for PacC processing, whereby PacC processing was drastically impaired in ΔpalH mutants at all pHs tested. The ΔpalH mutants were sensitive to alkaline pH and to cationic stress, whereby growth was completely abolished at pH 8.0 in the presence of 100 mM NaCl. The mutants were also sensitive to cell wall-perturbing agents such as 0.007% SDS and 20 mg/l congo red and in the presence of the cell wall-active antifungal caspofungin undergo extensive hyphal branching and ballooning compared to the parental and reconstituted strains. In the absence of PalH A. fumigatus-mediated damage to epithelial cells is significantly reduced in vitro. Furthermore, the ΔpalH mutants are drastically attenuated for virulence in a leukopenic murine model of pulmonary aspergillosis. Expression of the recombinant PalH and PalF proteins in Saccharomyces cerevisiae indicated stable interaction between the receptor and its cognate arrestin, and also indicated oligomerisation of the PalH receptor, a hypothesis supported by analysis of differentially-tagged PalH proteins. Iterative screening of the library of recombinant A. fumigatus preys identified several clones expressing putative PalH interactors. Concordant with a role for these gene products in pH-mediated signaling, a variety of pH-sensitive phenotypes were identified amongst the cohort of null mutants.
Our results indicate that PalH is an oligomerising, non-redundant activator of PacC-mediated signaling for adaptation to extracellular pH. This highly conserved fungal pH sensor is required for A. fumigatus virulence and echinocandin tolerance. Future studies will focus upon the mechanism of PalH-mediated pH sensing.