Rhizopus oryzae (heterotypic synonym: R. arrhizus) intrinsic voriconazole and fluconazole resistance has been linked to its CYP51A gene. However, the amino acid residues involved in this phenotype have not yet been established. A comparison between R. oryzae and Aspergillus fumigatus Cyp51Ap sequences showed differences in several amino acid residues. Some of them were already linked with voriconazole resistance in A. fumigatus. The objective of this work was to analyze the role of two natural polymorphisms in the intrinsic voriconazole resistance phenotype of R. oryzae (Y129F and T290A, equivalent to Y121F and T289A seen in triazole-resistant A. fumigatus). We have generated A. fumigatus chimeric strains harboring different R. oryzae CYP51A genes (wild-type and mutants). These mutant R. oryzae CYP51A genes were designed to carry nucleotide changes that produce mutations at Cyp51Ap residues 129 and 290 (emulating the Cyp51Ap protein of azole susceptible A. fumigatus). Antifungal susceptibilities were evaluated for all the obtained mutants. The polymorphism T290A (alone or in combination with Y129F) had no impact on triazole MIC. On the other hand, a > 8-fold decrease in voriconazole MICs was observed in A. fumigatus chimeric strains harboring the RoCYP51Ap-F129Y. This phenotype supports the assumption that the naturally occurring polymorphism Y129F at R. oryzae Cyp51Ap is responsible for its voriconazole resistance phenotype. In addition, these chimeric mutants were posaconazole hypersusceptible. Thus, our experimental data demonstrate that the RoCYP51Ap-F129 residue strongly impacts VRC susceptibility and that it would be related with posaconazole-RoCYP51Ap interaction. Lay summary Rhizopus oryzae is intrinsically resistant to voriconazole, a commonly used antifungal agent. In this work, we analyze the role of two natural polymorphisms present in the target of azole drugs. We established that F129 residue is responsible of the intrinsic voriconazole resistance in this species.

中文翻译：

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米根霉（R. arrhizus）Cyp51Ap 中天然存在的 Y129F 多态性解释了其固有的伏立康唑抗性

Rhizopus oryzae（异型同义词：R. arrhizus）内在的伏立康唑和氟康唑耐药性与其 CYP51A 基因有关。然而，该表型所涉及的氨基酸残基尚未确定。R. oryzae 和 Aspergillus fumigatus Cyp51Ap 序列之间的比较显示了几个氨基酸残基的差异。其中一些已经与烟曲霉对伏立康唑的耐药性有关。这项工作的目的是分析两个天然多态性在米曲霉内在伏立康唑抗性表型中的作用（Y129F 和 T290A，相当于在三唑抗性烟曲霉中看到的 Y121F 和 T289A）。我们已经产生了含有不同米根霉 CYP51A 基因（野生型和突变体）的烟曲霉嵌合菌株。这些突变体 R. oryzae CYP51A 基因被设计为携带在 Cyp51Ap 残基 129 和 290 处产生突变的核苷酸变化（模拟对唑类敏感的烟曲霉的 Cyp51Ap 蛋白）。评估所有获得的突变体的抗真菌敏感性。多态性 T290A（单独或与 Y129F 组合）对三唑 MIC 没有影响。另一方面，a > 在含有 RoCYP51Ap-F129Y 的烟曲霉嵌合菌株中观察到伏立康唑 MIC 降低了 8 倍。该表型支持这样的假设，即米根霉 Cyp51Ap 中天然存在的多态性 Y129F 是其伏立康唑耐药表型的原因。此外，这些嵌合突变体对泊沙康唑过敏。因此，我们的实验数据表明，RoCYP51Ap-F129 残基强烈影响 VRC 易感性，并且与泊沙康唑-RoCYP51Ap 相互作用有关。总结 米根霉对伏立康唑（一种常用的抗真菌剂）具有内在抗性。在这项工作中，我们分析了唑类药物靶标中存在的两种天然多态性的作用。我们确定 F129 残基是该物种固有的伏立康唑抗性的原因。