Sterol Biosynthesis and Azole Tolerance Is Governed by the Opposing Actions of SrbA and the CCAAT Binding Complex

Abstract

Azole drugs selectively target fungal sterol biosynthesis and are critical to our antifungal therapeutic arsenal. However, resistance to this class of drugs, particularly in the major human mould pathogen Aspergillus fumigatus, is emerging and reaching levels that have prompted some to suggest that there is a realistic probability that they will be lost for clinical use. The dominating class of pan-azole resistant isolates is characterized by the presence of a tandem repeat of at least 34 bases (TR34) within the promoter of cyp51A, the gene encoding the azole drug target sterol C14-demethylase. Here we demonstrate that the repeat sequence in TR34 is bound by both the sterol regulatory element binding protein (SREBP) SrbA, and the CCAAT binding complex (CBC). We show that the 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-CoA-reductase and sterol C14-demethylase. In agreement with these findings, inactivation of the CBC increased tolerance to different classes of drugs targeting ergosterol biosynthesis including the azoles, allylamines (terbinafine) and statins (simvastatin). We reveal that a clinically relevant mutation in HapE (P88L) significantly impairs the binding affinity of the CBC to its target site. 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 and systemic model of aspergillosis.

In Vivo Selection of a Unique Tandem Repeat Mediated Azole Resistance Mechanism (TR120) inAspergillus fumigatus cyp51A, Denmark

“…The F46Y/M172V/E427K substitutions in Cyp51A, found in both susceptible and resistant isolates, have been suggested to play no role or only a minor role in reduced azole susceptibilities ( 12 , 13 ). TRs in the promoter region of cyp51A have previously been linked to increased cyp51A gene expression and MICs because of duplicated srbA transcription factor binding motifs (SRE1 and SRE2) leading to increased expression of cyp51A ( 14 , 15 ). Taken together, our data suggest that TR 120 alone is an important driver of pan-azole resistance at a level comparable to that known to be mediated by the TR 34 /L98H mechanism.…”

Clinical implications of globally emerging azole resistance in Aspergillus fumigatus

“…It needs to be emphasized that, in comparision to the in-host development of azole resistance, the A. fumigatus strains from the environment have, in addition to the mutations in the cyp51A gene, a tandom repeat (TR) duplication in the promoter region. The presence of the TR repeat has been shown to drive overexpression of cyp51A by increasing the binding activity of the sterol regulatory element binding protein resulting in increased tolerance of azoles [35]. To date, three resistance mechanisms involving promotor duplications (TR 34 /L98H, TR 53 and TR46/Y121F/T289A) and single mutations (G54 and M220) have been found in A. fumigatus isolates from soil and air samples and in clinical specimens of patients (table 1) [24,[60][61][62].…”

A sphingolipid synthesis‐related protein OrmA in Aspergillus fumigatus is responsible for azole susceptibility and virulence

“…Moreover, it is postulated that ergosterols and sphingolipids may have a close physical relationship with the plasma membrane structure. Until now, the fungal ergosterol synthase pathway, including a series of catalytic enzymes and their regulators, such as the Dap family and SrbA, have been verified and reported (Gsaller et al, 2016a(Gsaller et al, , 2016bJ. Song et al, 2016).…”