Uncovering the molecular basis of cell membrane integrity in azole-susceptible and azole-resistant isolates of Aspergillus fumigatus

Author:

Martin Weichert (NL)

Abstract:

Background:
The azole class of antifungal compounds, which blocks ergosterol biosynthesis, is widely used to inhibit fungal growth. Since ergosterol is crucial for the properties and functions of fungal cell membranes, azoles cause membrane stress. Evidence indicates that the intense use of agricultural azole fungicides, which can enter the natural niches of Aspergillus fumigatus, selects several resistance mutations in the cyp51A gene that encodes the azole target enzyme. These mutations confer cross-resistance to medical azole drugs, thereby reducing the treatment options for Aspergillosis patients infected with azole-resistant environmental isolates. Although antimicrobial resistance is typically associated with fitness penalties, growth and pathogenicity of these azole-resistant isolates of A. fumigatus are normal, suggesting that additional mutations and/or molecular mechanisms compensate for possible fitness costs. Here, we hypothesize that mechanisms of cell membrane integrity contribute to the normal fitness of azole-resistant isolates. Based on previous studies on the role of conserved penta-EF (PEF)-hand proteins in promoting membrane integrity in the moulds, Neurospora crassa and Botrytis cinerea, we explore whether the orthologous PefA protein and related molecular factors in A. fumigatus facilitate the adaptation to membrane stress caused by azoles and other antifungal compounds.
Methods:
To functionally characterize PefA in A. fumigatus, we are employing CRISPR/Cas9-mediated gene editing tools to create ΔpefA deletion mutants and reporter strains with fluorescently tagged PefA constructs. Since direct membrane disruption caused by the ergosterol-binding polyenes and saponins activates the PEF-hand protein in N. crassa, we compare the growth of the ΔpefA mutants exposed to these antifungal compounds with azole-treated cultures. To phenotypically assess membrane stress responses in azole-susceptible and azole-resistant isolates, we are using a microtiter plate approach to measure the growth of nearly 100 strains from an environmental collection of A. fumigatus during exposure to membrane-disrupting compounds.
Results:
Consistent with results from N. crassa, the ΔpefA mutants of A. fumigatus showed impaired growth in the presence of the saponin, tomatine, but not when treated with the polyene, nystatin. Moreover, loss of PefA did not increase susceptibility to the azole, itraconazole. By live-cell imaging of a fluorescently-tagged PefA protein, we are now exploring the subcellular localization patterns of PefA during these different forms of membrane stress. Our initial phenotypic analysis of a subset of environmental isolates revealed a wide range of susceptibilities to tomatine and nystatin, prompting us to next compare the membrane stress response profiles in between strains with different cyp51A genotypes. Finally, we are testing whether these susceptibility and tolerance phenotypes are linked to PefA-mediated mechanisms of membrane integrity.
Conclusions:
Investigating membrane stress responses in A. fumigatus during the exposure to azoles and other antifungal compounds will broaden our understanding of the molecular basis of fungal cell membrane integrity. In addition, uncovering the heterogeneity of membrane stress adaptation in azole-susceptible and azole-resistant isolates might inform novel strategies against antifungal resistance in A. fumigatus and other pathogenic fungi.

Abstract Number: 10

Conference Year: 2024

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