Mechanistic Basis of pH-Dependent 5-Flucytosine Resistance in Aspergillus fumigatus

Abstract

The antifungal drug 5-flucytosine (5FC), a derivative of the nucleobase cytosine, is licensed for the treatment of fungal diseases; however, it is rarely used as a monotherapeutic to treat Aspergillus infection. Despite being potent against other fungal pathogens, 5FC has limited activity against Aspergillus fumigatus when standard in vitro assays are used to determine susceptibility. However, in modified in vitro assays where the pH is set to pH 5, the activity of 5FC increases significantly. Here we provide evidence that fcyB, a gene that encodes a purine-cytosine permease orthologous to known 5FC importers, is downregulated at pH 7 and is the primary factor responsible for the low efficacy of 5FC at pH 7. We also uncover two transcriptional regulators that are responsible for the repression of fcyB and, consequently, mediators of 5FC resistance, the CCAAT binding complex (CBC) and the pH regulatory protein PacC. We propose that the activity of 5FC might be enhanced by the perturbation of factors that repress fcyB expression, such as PacC or other components of the pH-sensing machinery.

Keywords: 5-flucytosine; Aspergillus fumigatus; CBC; antifungal agents; antifungal drug resistance; pH regulation; pacC; transcription factors.

FIG 1

PacC and the CBC regulate 5FC resistance via fcyB. Susceptibilities of mutant strains were determined according to the EUCAST broth microdilution reference method using RPMI (21) (A) and on AMM agar (B). For both experiments, isolates were incubated for 48 h at 37°C.

FIG 2

fcyB expression is repressed at neutral pH. The deletion of either hapC or pacC leads to the transcriptional upregulation of fcyB. Low pH results in a significant increase in fcyB transcript levels in the wild type. In contrast to the mutation of the CBC (ΔhapC), the ΔpacC mutant also shows a significant upregulation of the gene at low pH. n.s., not significant.

FIG 3

The CBC directly interacts with the fcyB promoter in vivo. (A) ChIP-seq peaks (20) with putative CBC binding regions (red, CCAAT boxes) are located at positions −663 and −568 relative to the translation start (TLS). (B) Based on the peak summit (position −620 relative to the TLS), direct in vivo binding of the CBC to the promoter of fcyB was validated by employing ChIP-qPCR (see Materials and Methods).

FIG 4

Overexpression of fcyB results in pH-independent 5FC hypersusceptibility. (A) For the conditional overexpression of fcyB, its native promoter region (wt) was replaced by the PxylP-based, xylose-inducible promoter system (fcyB^XYL). (B) AMM was supplemented with 1% xylose (i., induced) to overexpress fcyB. To downregulate the expression of the gene in the fcyB^XYL strain, xylose was omitted (n.i., noninduced). (C) MIC levels under inducing and noninducing conditions were determined according to EUCAST guidelines (21). For both experiments, strains were incubated for 48 h at 37°C. *, P < 0.05.

FIG 5

Proposed regulatory mechanism determining pH-dependent 5FC activity in A. fumigatus. The major route of 5FC entry into A. fumigatus cells occurs via uptake through the purine-cytosine permease FcyB. fcyB gene expression is repressed at high pH but transcriptionally upregulated at low pH. At pH 7, fcyB is repressed by the CBC and PacC, which confers intrinsic resistance to 5FC. The CBC acts as a direct repressor of the gene. In contrast to the CBC, PacC also exerts a negative regulatory function on fcyB gene expression at low pH.