A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations - PubMed (original) (raw)

A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations

E Mellado et al. Antimicrob Agents Chemother. 2007 Jun.

Abstract

Fourteen Aspergillus fumigatus clinical isolates that exhibited a pattern of reduced susceptibility to triazole drugs were analyzed. The sequences of the cyp51A gene from all isolates showed the presence of a point mutation at t364a, which led to the substitution of leucine 98 for histidine (L98H), together with the presence of two copies of a 34-bp sequence in tandem in the promoter of the cyp51A gene. Quantitative expression analysis (real-time PCR) showed up to an eightfold increase in the level of expression of the cyp51A gene compared to that by the susceptible strain. Three PCR fragments of one azole-resistant strain (strain CM2627) that included the promoter with the tandem repeat and part of cyp51A with the t364a mutation or PCR fragments with only one of the modifications were used to replace the cyp51A gene of an azole drug-susceptible A. fumigatus wild-type strain (strain CM237). Only transformants which had incorporated the tandem repeat in the promoter of the cyp51A gene and the L98H amino acid substitution exhibited similarly reduced patterns of susceptibility to all triazole agents and similarly increased levels of cyp51A expression, confirming that the combination of both alterations was responsible for the azole-resistant phenotype.

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Figures

FIG. 1.

FIG. 1.

Diagram of the A. fumigatus cyp51A gene and promoter showing the locations of the gene modifications and the primers used for PCR amplification of the different cyp51A gene fragments: (A) Wild-type strain CM237; (B) azole-resistant strain CM2627. The primer sets were as follows: (i) primer set A7-A2, which contains the TR and the t364a base change; (ii) primer set A7-A2, which contains only the 34-bp TR; and (iii) primer set P450A1-P450A2, which amplifies the full cyp51A gene, including only the t364a base change (L98H).

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