Toepfer Stephanie, Lackner Michaela, Keniya Mikhail V, Monk Brian C
Faculty of Dentistry, Sir John Walsh Research Institute, University of Otago, Dunedin 9016, New Zealand.
Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Schöpfstrasse 41, 6020 Innsbruck, Austria.
J Fungi (Basel). 2023 Jan 27;9(2):168. doi: 10.3390/jof9020168.
infections are difficult to treat due to acquired drug resistance against one or multiple antifungal drug classes. The most prominent resistance mechanisms in are overexpression and point mutations in Erg11, and the overexpression of efflux pump genes and . We report the establishment of a novel platform for molecular analysis and drug screening based on acquired azole-resistance mechanisms found in . Constitutive functional overexpression of wild-type Erg11, Erg11 with amino acid substitutions Y132F or K143R and the recombinant efflux pumps Cdr1 and Mdr1 has been achieved in . Phenotypes were evaluated for standard azoles and the tetrazole VT-1161. Overexpression of CauErg11 Y132F, CauErg11 K143R, and CauMdr1 conferred resistance exclusively to the short-tailed azoles Fluconazole and Voriconazole. Strains overexpressing the Cdr1 protein were pan-azole resistant. While CauErg11 Y132F increased VT-1161 resistance, K143R had no impact. Type II binding spectra showed tight azole binding to the affinity-purified recombinant CauErg11 protein. The Nile Red assay confirmed the efflux functions of CauMdr1 and CauCdr1, which were specifically inhibited by MCC1189 and Beauvericin, respectively. CauCdr1 exhibited ATPase activity that was inhibited by Oligomycin. The overexpression platform enables evaluation of the interaction of existing and novel azole drugs with their primary target CauErg11 and their susceptibility to drug efflux.
由于对一种或多种抗真菌药物类别产生获得性耐药，感染难以治疗。[具体物种]中最突出的耐药机制是Erg11的过表达和点突变，以及外排泵基因[具体基因名称1]和[具体基因名称2]的过表达。我们报告了基于在[具体物种]中发现的获得性唑类耐药机制建立了一个用于分子分析和药物筛选的新平台。在[具体物种]中实现了野生型[具体物种]Erg11、具有氨基酸取代Y132F或K143R的Erg11以及重组外排泵Cdr1和Mdr1的组成型功能过表达。对标准唑类药物和四氮唑VT - 1161的表型进行了评估。CauErg11 Y132F、CauErg11 K143R和CauMdr1的过表达仅赋予对短尾唑类氟康唑和伏立康唑的耐药性。过表达Cdr1蛋白的菌株对所有唑类药物耐药。虽然CauErg11 Y132F增加了VT - 1161耐药性，但K143R没有影响。II型结合光谱显示唑类药物与亲和纯化的重组CauErg11蛋白紧密结合。尼罗红测定法证实了CauMdr1和CauCdr1的外排功能，它们分别被MCC1189和白僵菌素特异性抑制。CauCdr1表现出被寡霉素抑制的ATP酶活性。[具体物种]过表达平台能够评估现有和新型唑类药物与其主要靶点CauErg11的相互作用以及它们对药物外排的敏感性。
