Identification of Two Different 14-α Sterol Demethylase-Related Genes ( cyp51A and cyp51B ) in Aspergillus fumigatus and Other Aspergillus species

Abstract

Two cyp51-related genes (cyp51A and cyp51B) encoding 14-␣ sterol demethylase-like enzymes were identified in the opportunistic human pathogen Aspergillus fumigatus. PCR amplification using degenerate oligonucleotides based on conserved areas of cytochrome P450 demethylases of other filamentous fungi and yeasts allowed the cloning and sequencing of two different homologue genes in A. fumigatus. Southern analysis confirmed that both genes hybridized to distinct genomic loci and that both are represented as single copies in the genome. Comparison of the deduced Cyp51A and Cyp51B proteins with the CYP51 proteins from Penicillium italicum, Aspergillus nidulans, Erysiphe graminis, Uncinula necator, Botrytis cinerea, Ustilago maydis, Cryptococcus neoformans, Candida albicans, Saccharomyces cerevisiae, Candida tropicalis, and Candida glabrata showed that the percentages of identity of the amino acid sequences (range, 40 to 70%) were high enough to consider Cyp51A and Cyp51B to be members of the fungal CYP51 family. Fragments from both genes were also cloned from other Aspergillus spp. (A. flavus, A. nidulans, and A. terreus). Phylogenetic analysis showed that, at least in the most pathogenic species of Aspergillus, there are two fungal CYP51 proteins. This is the first report of the existence of two homologue genes coding for 14-␣ sterol demethylase in the fungal kingdom. This finding could provide insights into the azole resistance mechanisms operating in fungi. The primers used here may be useful molecular tools for facilitating the cloning of novel 14-␣ sterol demethylase genes in other filamentous fungi.

Rapid Detection of Triazole Antifungal Resistance in Aspergillus fumigatus

“…Eleven strains belong to the Public Health Research Institute (PHRI) collection, eight of them were A. fumigatus clinical isolates (kindly provided by D. Denning, Manchester University, Manchester, United Kingdom) (18) and three laboratory-derived mutants (33). Moreover, 10 A. fumigatus with known cyp51A mutations (G54V, G54E, G54R, G54W, TR-L98H, G138C, M220V, M220K, M220T, and M220I) (17,27) or isolated with the Q-Biogene FastDNA kit (Irvine, CA) according to the manufacturer's instructions.…”

“…fumigatus cyp51A gene DNA sequencing. The full coding sequences and promoter (Ϫ500 from the start codon) of cyp51A were amplified and sequenced in both directions, as previously described (27). In Spain, DNA sequencing was performed by using a BigDye terminator cycle sequencing ready-reaction system (Applied Biosystems, Madrid, Spain) according to the manufacturer's instructions.…”

“…In A. fumigatus there are two distinct but related 14-␣ sterol demethylase (cyp51) proteins encoded by cyp51A and cyp51B (27), although only mutations in Cyp51A are important for clinical resistance. These mutations confer several different susceptibility profiles: (i) resistance to ITC and PSC associated with amino acid substitutions at Cyp51A glycine 54 (Gly 54) (9,23,33); (ii) a pattern of resistance to ITC and high VRC, RVC, and PSC MICs (strains exhibiting this susceptibility profile harbor amino acid substitutions at methionine 220 [Met 220]) (3,28); (iii) a pattern of azole cross-resistance associated with higher cyp51A expression produced by a tandem repeat (TR) of a 34-bp sequence in the cyp51A gene promoter in combination with an amino acid substitution at Cyp51A leucine 98 (TR-L98H) (29,44); and (iv) a triazole cross-resistance related to an amino acid change at Cyp51A glycine 138 to cysteine (G138C) (18).…”

Determination of Azole Antifungal Drug Resistance Mechanisms Involving Cyp51a Gene in Clinical Isolates of Aspergillus fumigatus and Aspergillus niger

“…The inoculum suspension of the fungal conidia was prepared at 0.4 × 10 ³ CFU/ml to 0.5 ×10 ³ CFU/ml using spectrophotometer (530 nm) (OD; 0.09-0.13) as described in CLSI (formerly NCCLS) document M38-A (33) [9]. Susceptibility assay was performed by the E-test method according to the manufacturer's instructions (AB Biodisk, Sweden).…”

Rapid, High-Throughput, Multiplex, Real-Time PCR for Identification of Mutations in the cyp51A Gene of Aspergillus fumigatus That Confer Resistance to Itraconazole

“…Itraconazole resistance in clinical isolates as well as in laboratory mutant strains has been reported (3,5,18,24,32). Triazole drugs bind to the active site of the fungal cytochome P450 14-␣-sterol demethylase, which catalyzes the 14␣ demethylation of ergosterol precursors (22) and which is encoded by cyp51A in Aspergillus (29). Several amino acid substitutions in Cyp51A resulting in itraconazole resistance in A. fumigatus have been described (7,26,33).…”