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

Abstract

29The antifungal drug 5-flucytosine (5FC), a derivative of the nucleobase cytosine, is 30 licenced for treatment of fungal diseases however it is rarely used as a monotherapeutic 31 to treat Aspergillus infection. Despite being potent against other fungal pathogens, 5FC 32 has limited activity against A. fumigatus when standard in vitro assays are used to 33 determine susceptibility. However, in modified in vitro assays where the pH is set to pH 34 5 the activity of 5FC increases significantly. 35Here we provide evidence that fcyB, a gene that encodes a purine-cytosine permease 36 orthologous to known 5FC importers is downregulated at pH 7 and is the primary factor 37 responsible for the low efficacy of 5FC at pH 7. We also uncover two transcriptional 38 regulators that are responsible for repression of fcyB and consequently mediators of 5FC 39 resistance, the CCAAT binding complex (CBC) and the pH regulatory protein PacC. We 40propose that the activity of 5FC might be enhanced by perturbation of factors that 41repress fcyB expression such as PacC or other components of the pH sensing machinery.

Multiplex genetic engineering exploiting pyrimidine salvage pathway-based self-encoded selectable markers

“…Plate growth-based susceptibility testing revealed that, similar to previous work (Gsaller et al 2018), 5FC levels ≥ 10 μg/ml blocked wt growth at pH5, while 100 μg/ml 5FC were required at pH7 (Fig. 1b).…”

“…Inactivation of each of these steps resulted in 5FC resistance, whereby inactivation of Fur1p also conferred 5FU resistance (Kern et al 1990). Orthologous proteins from S. cerevisiae (Fcy2p), A. fumigatus and Aspergillus nidulans (FcyB), respectively, have been identified as major cellular 5FC importers (Paluszynski et al 2006; Vlanti and Diallinas 2008; Gsaller et al 2018).…”

“…To analyze their role in 5FC as well as 5FU metabolism, we inactivated fcyA and uprt in the A. fumigatus strain A1160P+ (Fraczek et al 2013), termed wt here, using hygromycin and phleomycin resistance-based deletion cassettes. Due to the interdependency of 5FC activity and environmental pH (Te Dorsthorst et al 2004; Gsaller et al 2018), we investigated the contribution of both enzymes, as well as FcyB, to 5FC and 5FU activity at both pH5 and pH7.…”

“…For the regeneration of transformants, solid AMM ( A. fumigatus and P. chrysogenum) or PDA ( F. oxysporum) supplemented with 342 g/l or 200 g/l sucrose, respectively, were used. Selection procedures using conventional selectable marker genes ( hph , ble ) were carried out as described previously for A. fumigatus (Gsaller et al 2018).…”

Resistance in the Environmental Pathogenic Fungus <i>Aspergillus fumigatus</i>

“…The repeat is 34-, 46-, or 53-bp long and located approximately 300 bp upstream of the cyp51A start codon. The region contains AtrR-and SrbA-binding elements [6][7][8] , which regulate cyp51A expression. In addition to the duplication of the region upregulating cyp51A expression, amino acid substitutions contribute to azole resistance, except in the TR53-type strain, which contains no substitution in cyp51A.…”