Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.
Mycology Reference Laboratory, National Centre for Microbiology, Instituto de Salud Carlos III (ISCIII), Madrid, Spain.
PLoS Pathog. 2022 Dec 27;18(12):e1011066. doi: 10.1371/journal.ppat.1011066. eCollection 2022 Dec.
Invasive aspergillosis remains one of the most devastating fungal diseases and is predominantly linked to infections caused by the opportunistic human mold pathogen Aspergillus fumigatus. Major treatment regimens for the disease comprise the administration of antifungals belonging to the azole, polyene and echinocandin drug class. The prodrug 5-fluorocytosine (5FC), which is the only representative of a fourth class, the nucleobase analogs, shows unsatisfactory in vitro activities and is barely used for the treatment of aspergillosis. The main route of 5FC activation in A. fumigatus comprises its deamination into 5-fluorouracil (5FU) by FcyA, which is followed by Uprt-mediated 5FU phosphoribosylation into 5-fluorouridine monophosphate (5FUMP). In this study, we characterized and examined the role of a metabolic bypass that generates this nucleotide via 5-fluorouridine (5FUR) through uridine phosphorylase and uridine kinase activities. Resistance profiling of mutants lacking distinct pyrimidine salvage activities suggested a minor contribution of the alternative route in 5FUMP formation. We further analyzed the contribution of drug efflux in 5FC tolerance and found that A. fumigatus cells exposed to 5FC reduce intracellular fluoropyrimidine levels through their export into the environment. This release, which was particularly high in mutants lacking Uprt, generates a toxic environment for cytosine deaminase lacking mutants as well as mammalian cells. Employing the broad-spectrum fungal efflux pump inhibitor clorgyline, we demonstrate synergistic properties of this compound in combination with 5FC, 5FU as well as 5FUR.
侵袭性曲霉病仍然是最具破坏性的真菌病之一，主要与机会性人类霉菌病原体烟曲霉引起的感染有关。该疾病的主要治疗方案包括使用属于唑类、多烯类和棘白菌素类药物类别的抗真菌药物。前药 5-氟胞嘧啶（5FC）是第四类核苷类似物的唯一代表，其体外活性不佳，几乎不用于曲霉病的治疗。5FC 在烟曲霉中的主要激活途径包括其被 FcyA 脱氨为 5-氟尿嘧啶（5FU），随后由 Uprt 介导将 5FU 磷酸核糖化为 5-氟尿嘧啶单磷酸（5FUMP）。在这项研究中，我们对通过尿苷磷酸化酶和尿苷激酶活性生成该核苷酸的代谢旁路进行了表征和研究。缺乏不同嘧啶补救活性的突变体的耐药性分析表明，该替代途径在 5FUMP 形成中的作用较小。我们进一步分析了药物外排在 5FC 耐受性中的作用，发现暴露于 5FC 的烟曲霉细胞通过将其排出到环境中来降低细胞内氟嘧啶水平。这种释放在缺乏 Uprt 的突变体中特别高，会为缺乏胞嘧啶脱氨酶的突变体以及哺乳动物细胞产生毒性环境。我们使用广谱真菌外排泵抑制剂氯胍，证明了该化合物与 5FC、5FU 和 5FUR 联合使用具有协同作用。
