The molecular basis of intrinsic resistance to azoles in Rhizopus arrhizus

Abstract

The fungal disease mucormycosis, while generally regarded as rare and not transmitted between individuals, has become increasingly prevalent in disaster areas, among the immunocompromised, and in diabetics especially in response to COVID-19. Treatment options are limited. These include debridement of necrotizing tissue followed by complicated multicomponent therapies with amphotericin B and selected azole drugs, usually having poor outcomes. Mucormycetes are intrinsically resistant to the widely used short-tailed azole drugs fluconazole and voriconazole, but susceptible to the long-tailed, though expensive, azole posaconazole. Knowledge of the crystal structure of Saccharomyces cerevisiae sterol 14α-demethylase (Erg11, Cyp51) led to the hypothesis that this pattern of intrinsic azole resistance and susceptibility is due to the Rhizopus arrhizus CYP51-F5 isoform residues F129 and A291, while the CYP51-F1 isoform residues Y127 and V291 confer susceptibility to both short- and long-tailed azole drugs. The heterologous overexpression of individual recombinant R. arrhizus CYP51 isoforms in a S. cerevisiae host, with or without the cognate NADPH-cytochrome P450 reductase (RaCPR), and selective genetic modification of CYP51-F5 have tested this hypothesis. Complementary gene deletion experiments in Rhizopus microsporus confirm that the amino acid residues that align with R. arrhizus CYP51-F5 F129 and A291 determine the resistance or susceptibility pattern of R. arrhizus to short-, medium-, and long-tailed azoles.