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

Abstract

Rhizopus oryzae is the most prevalent causative agent of mucormycosis, an increasingly reported opportunistic fungal infection. These Mucorales are intrinsically resistant to Candida- and Aspergillus-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 Rhizopus oryzaeCYP51 genes are uniquely responsible for intrinsic voriconazole and fluconazole resistance in these fungal pathogens. Two CYP51 genes were identified in the R. oryzae genome. We classified them as CYP51A and CYP51B based on their sequence similarity with other known fungal CYP51 genes. Later, we obtained a chimeric Aspergillus fumigatus strain harboring a functional R. oryzae CYP51A gene expressed under the regulation of the wild-type A. fumigatusCYP51A promoter and terminator. The mutant was selected after transformation by using a novel procedure taking advantage of the FLC hypersusceptibility of the A. fumigatusCYP51A deletion mutant used as the recipient strain. The azole susceptibility patterns of the A. fumigatus transformants harboring R. oryzae CYP51A mimicked exactly the azole susceptibility patterns of this mucormycete. The data presented in this work demonstrate that the R. oryzae CYP51A coding sequence is uniquely responsible for the R. oryzae azole susceptibility patterns.

Keywords: CYP51; Mucorales; Rhizopus; azole; fluconazole; intrinsic resistance; molecular mechanism; resistance; voriconazole.

FIG 1

Agarose gel (1.5% agarose) electrophoresis at 40 V for 3 h showing the detection of R. oryzae CYP51 transcripts using genomic DNA (lanes 1 and 3) and cDNA (lanes 2 and 4) as the templates. Lanes 1 and 2, amplification of RoCYP51A (RO3G_16595) 1,626-nt and 1,524-nt bands, respectively; lanes 3 and 4, the resolved PCR bands of RoCYP51B (RO3G_11790) at 1,755 nt and 1,533 nt, respectively.

FIG 2

Neighbor-joining phylogenetic tree of 55 different fungal 14-α sterol demethylases, including enzymes from 9 Ascomycetes yeast species, 20 Ascomycetes mold species, 9 Basidiomycetes species, and 7 Mucorales species. GenBank accession numbers appear after the species names. Some fungal genus names were abbreviated as follows to esthetically improve the figure: A., Aspergillus; S., Saccharomyces; C., Candida; F., Fusarium; R., Rhizopus.

FIG 3

(A, B) Selection plates using FLC (100 μg/ml) where LMDM-1229 (A) and LMDM-1230 (B) transformants were obtained. (C, D) Replica plates used minimal medium (C) and minimal medium with 450 μg/ml hygromycin B (D).

FIG 4

(A) Scheme of the LMDM-p134 vector generation process used in this work. Striped and black boxes represent the 5′ and 3′ UTRs of the AfCYP51A included and not included in the transformation vector, respectively. Light gray boxes denote the RoCYP51A (RO3G_16595) complete coding sequence obtained from cDNA. Dark gray boxes show the C- and N-terminal fractions of AfCYP51A (12, 32). Thin lines in LMDM-p125 and LMDM-p134 symbolize the pGEM-T Easy vector. Arrows represent primers, and their coloring shows where they hybridized to each of the genomic or vector DNAs. (B) Integration and homologous recombination confirmation by using PCRs. Lane M, 100-nt ladder (numbers on the left are in nucleotides); lanes 1 to 4, RoCYP51A (RO3G_16595) exon 3 amplifications using the P-RoCYP51F3 and P-RoCYP51R3 primers (1,374 nt); lanes 5 to 8, multiplex PCR with the A13, A5R and P-RoCYP51R1 primers to confirm homologous recombination (by the presence of the 1,286-nt band) or the lack of existence of RoCYP51A (RO3G_16595) in the A. fumigatus genome (by the presence of the 1,461-nt band); lane 1, LMDM-p134; lanes 2 and 5, R. oryzae ATCC 11886; lanes 3 and 6, A. fumigatus LMDM-1229; lane 7, A. fumigatus LMDM-1230; lanes 4 and 8, A. fumigatus LMDM-32.

FIG 5

Disk diffusion susceptibility testing using fluconazole (FLC) and voriconazole (VRC) paper disks for LMDM-31 (parental strain akuB_KU80Δ) (31), LMDM-32 (recipient strain akuB_KU80Δ CYP51AΔ) (12), R. oryzae ATCC 11886, and A. fumigatus chimera LMDM-1229 (akuB_KU80Δ RoCYP51A).