Intrinsic resistance to short-tailed azoles in the basal fungus Mucor lusitanicus: functional analysis of Cyp51 isoforms and amino acid substitutions

Abstract

Introduction: The basal fungus Mucor lusitanicus (Mlu) is a cause of mucormycosis, with limited treatment options due to intrinsic resistance to short-tailed azoles (fluconazole, voriconazole) and echinocandins. In silico analysis identified amino-acid-substitutions Y129F and V293A in the substrate-binding pocket of sterol-14α-demethylase (Cyp51, Erg11) isoform F5 as potential resistance mechanisms. Methods: This hypothesis has been experimentally tested for M. lusitanicus by expressing its Cyp51 isoforms (MluCyp51-F1, MluCyp51-F5) and modified MluCyp51-F5 variants (F129Y, A293V, F129Y A293V) with/without their cognate NADPH-cytochrome-P450-reductase (MluCPR) in Saccharomyces cerevisiae. Results: Strains expressing MluCyp51 isoforms +/− MluCPR showed expression levels of 38–69% compared to an overexpressed Erg11 control. Susceptibility assays confirmed that MluCyp51-F5 confers intrinsic resistance to short-tailed azoles, while substitutions F129Y, A293V, or both restored susceptibility. Growth- and susceptibility-assays revealed that the MluCyp51-F1 + CPR construct had a voriconazole MIC of 0.5 μM, while MluCyp51-F5 + CPR had a MIC of 11.5 μM, with no changes in growth rate or ergosterol composition at 1.0 μM voriconazole. Susceptibility to long-tailed azoles (e.g., posaconazole) remained unchanged for both isoforms. Discussion: These findings demonstrate the functional expression of MluCyp51-F1 and MluCyp51-F5 isoforms in a phylogenetically distant host and confirm that conserved substitutions Y129F and V293A in MluCyp51-F5 confer intrinsic resistance to short-tailed azoles in M. lusitanicus.