Antifungal azole drugs have been the cornerstone of systemic antifungal therapy for the last 30 years. However, resistance to the azoles, particularly in the major human mould pathogen Aspergillus fumigatus, is emerging and reaching levels that have prompted some centres to move away from azoles as a sole birst line therapeutic. One particular family of pan-azole resistant isolates dominates. Strains with TR34/L98H, a combination of a 34 base pair tandem repeat in the cyp51A 5’ non-translated region with a mutation of the coding sequence resulting in the amino acid alteration L98H, have been found globally. Isolates harboring the TR34 mutant allele display increased cyp51A expression levels however, the precise mechanism underlying upregulation of the gene remained unclear. Two transcriptional regulators have been associated with modibied azole tolerance in A. fumigatus, the sterol regulatory element SrbA, and the CCAAT binding complex (CBC). SrbA acts a positive regulator of ergosterol biosynthesis and promotes azole tolerance. Hence loss of SrbA activity results in an increase in azole susceptibility. Modibication of the HapE subunit of the CBC (to HapEP88L) has been shown promote azole resistance. Here we demonstrate that the 34 mer duplicated in TR34 is bound by both SrbA and the CBC. We show that CBC acts complementary to SrbA as a negative regulator of ergosterol biosynthesis and show that lack of CBC activity results in increased sterol levels via transcriptional derepression of multiple ergosterol biosynthetic genes including those coding for HMG-CoA-synthase, HMG-CoAreductase and sterol 14-alpha demethylase. We reveal that the P88L substitution within HapE signibicantly impairs the binding afbinity of the CBC to its target site resulting in derepression of cyp51A and other genes in the ergosterol
biosynthetic pathway. We identify that the mechanism underpinning TR34 driven overexpression of cyp51A results from duplication of SrbA but not CBC binding sites and show that deletion of the 34 mer results in lack of cyp51A expression and increased azole susceptibility similar to a cyp51A null mutant. Finally we show that strains lacking a functional CBC are severely attenuated for pathogenicity in a pulmonary systemic model of aspergillosis indicating that, although null mutants are resistant to azoles, they would be unlikely to be able to sustain an infection.
Full conference title:
- Asperfest 13 (2016)