Molecular Confirmation of the Linkage between the <i>Rhizopus oryzae CYP51A</i> Gene Coding Region and Its Intrinsic Voriconazole and Fluconazole Resistance

Abstract

<i>Rhizopus oryzae</i> is the most prevalent causative agent of mucormycosis, an increasingly reported opportunistic fungal infection. These Mucorales are intrinsically resistant to <i>Candida</i>- and <i>Aspergillus</i>-active antifungal azole drugs, such as fluconazole (FLC) and voriconazole, respectively. Despite its importance, the molecular mechanisms of its intrinsic azole resistance have not been elucidated yet. The aim of this work was to establish if the <i>Rhizopus oryzae</i><i>CYP51</i> genes are uniquely responsible for intrinsic voriconazole and fluconazole resistance in these fungal pathogens. Two <i>CYP51</i> genes were identified in the <i>R. oryzae</i> genome. We classified them as <i>CYP51A</i> and <i>CYP51B</i> based on their sequence similarity with other known fungal <i>CYP51</i> genes. Later, we obtained a chimeric <i>Aspergillus fumigatus</i> strain harboring a functional <i>R. oryzae CYP51A</i> gene expressed under the regulation of the wild-type <i>A. fumigatus</i><i>CYP51A</i> promoter and terminator. The mutant was selected after transformation by using a novel procedure taking advantage of the FLC hypersusceptibility of the <i>A. fumigatus</i><i>CYP51A</i> deletion mutant used as the recipient strain. The azole susceptibility patterns of the <i>A. fumigatus</i> transformants harboring <i>R. oryzae CYP51A</i> mimicked exactly the azole susceptibility patterns of this mucormycete. The data presented in this work demonstrate that the <i>R. oryzae CYP51A</i> coding sequence is uniquely responsible for the <i>R. oryzae</i> azole susceptibility patterns.