The ability to adapt to external pH is a fundamental requisite for the success of Aspergillus species in multiple niches being relevant to growth in the environment, and also in the infected host. An efficient response of Aspergillus species to extracellular pH flux requires the integrity of seven genes, including the DNA binding transcription factor PacC. Besides its role in adaptation to alkaline conditions, we found that A. fumigatus PacC is required for virulence in neutropenic mice.
To identify novel and crucial Rim101p/PacC regulators, a global phenotypic screen of the Euroscarf S. cerevisiae ORF deletion library was performed to identify mutants having identical cation hypersensitive phenotypes to 916;rim101, the S. cerevisiae homologue of PacC. An epistacy screen to address the impact of activated Rim101p upon cation sensitivity was performed, along with a screen for mutants aberrantly regulating a synthetic Rim101p-regulatable promoter. These analyses identified 10 mutants suffering aberrant Rim101p processing and/or transcriptional activity, including mutants deficient in the cell wall integrity regulators Bck1p and Slt2p/Mpk1p.
To identify gene functions under PacC regulatory control during early infection we performed transcriptional profiling upon an A. fumigatus ATCC46645 clinical isolate and an isogenic 916;pacC mutant using germlings extracted from bronchoalveolar lavage (BAL), and assessing transcript abundance, on a global scale, at 4, 8 and 16 hours post-infection. Aberrant transcription of multiple transporter and secondary metabolism functions was identified during the early time course of infection. A significant impact upon cell wall biosynthesis was also evident from this analysis, and accordingly, pH-non-sensing A fumigatus mutants were found to be highly susceptible to cell wall-active antifungal drugs.
In order to assess the role of the cell wall integrity pathway in pH adaptation we tested the phenotypes of A. fumigatus null mutants of Bck1 and Mpk1, finding pH-sensitive phenotypes for these mutants in vitro as well as sensitivity to cell wall damaging agents which was potentiated at alkaline pH. We therefore hypothesise that PacC acts downstream of the cell wall integrity pathway to direct the regulation of cell wall biosynthesis in accordance with environmental pH. Given that the A. fumigatus cell wall is essential for viability, agents which selectively inhibit the pH-dependent activation of PacC signalling might provide useful adjuncts to existing antifungal therapies.
Full conference title:
- AAA 5th (2012)