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Contributions of both ATP-Binding Cassette Transporter and Cyp51A Proteins Are Essential for Azole Resistance in
Sanjoy Paul1, Daniel Diekema2, W Scott Moye-Rowley3
1Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.
Abstract:
While azole drugs targeting the biosynthesis of ergosterol are effective antifungal agents, their extensive use has led to the development of resistant organisms. Infections involving azole-resistant forms of the filamentous fungus Aspergillus fumigatus are often associated with genetic changes in the cyp51A gene encoding the lanosterol α14 demethylase target enzyme. Both a sequence duplication in the cyp51A promoter (TR34) and a substitution mutation in the coding sequence (L98H) are required for the full expression of azole resistance. A mechanism commonly observed in pathogenic yeast such as Candida albicans involves gain-of-function mutations in transcriptional regulatory proteins that induce expression of genes encoding ATP-binding cassette (ABC) transporters. We and others have found that an ABC transporter protein called Cdr1B (here referred to as AbcG1) is required for wild-type azole resistance in A. fumigatus Here, we test the genetic relationship between the TR34 L98H allele of cyp51A and an abcG1 null mutation. Loss of AbcG1 from a TR34 L98H cyp51A-containing strain caused a large decrease in the azole resistance of the resulting double-mutant strain. We also generated antibodies that enabled the detection of both the wild-type and L98H forms of the Cyp51A protein. The introduction of the L98H lesion into the cyp51A gene led to a decreased production of immunoreactive enzyme, suggesting that this mutant protein is unstable. Our data confirm the importance of AbcG1 function during azole resistance even in a strongly drug-resistant background.
Insights
Azole resistance in Aspergillus fumigatus involves the Cyp51A enzyme and the AbcG1 transporter. Loss of AbcG1 significantly reduced resistance in azole-resistant strains, highlighting AbcG1
Area of Science:
- Medical Mycology
- Antifungal Drug Resistance
- Molecular Biology
Background:
- Azole antifungal drugs are crucial for treating fungal infections but face challenges due to emerging resistance.
- Azole resistance in *Aspergillus fumigatus* is often linked to mutations in the *cyp51A* gene, encoding lanosterol α14 demethylase.
- ATP-binding cassette (ABC) transporters, like AbcG1, play a role in antifungal resistance, a mechanism seen in other yeasts.
Purpose of the Study:
- To investigate the genetic interaction between the TR34 L98H *cyp51A* allele and an *abcG1* null mutation in *Aspergillus fumigatus*.
- To determine the contribution of the AbcG1 transporter to azole resistance in the context of a highly resistant *cyp51A* mutation.
Main Methods:
- Generated and analyzed a double-mutant strain lacking functional AbcG1 in a background with the TR34 L98H *cyp51A* allele.
- Assessed azole resistance levels in wild-type, single-mutant, and double-mutant strains.
- Developed antibodies to detect and quantify wild-type and L98H forms of the Cyp51A protein.
Main Results:
- Deletion of the *abcG1* gene in a TR34 L98H *cyp51A* strain significantly decreased azole resistance.
- The L98H mutation in *cyp51A* resulted in reduced production of immunoreactive Cyp51A protein, suggesting instability.
- The study confirms the essential role of AbcG1 in maintaining azole resistance, even in strains with strong pre-existing resistance mechanisms.
Conclusions:
- AbcG1 is a critical factor for azole resistance in *Aspergillus fumigatus*, independent of the *cyp51A* mutation status.
- The instability of the L98H Cyp51A mutant protein may influence the overall resistance phenotype.
- Targeting AbcG1 could be a potential strategy to overcome azole resistance in *Aspergillus fumigatus* infections.
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