RESUMEN
Real-time quantitative PCR was used to measure expression levels of genes encoding efflux pumps, ERG11 and two control genes, ACT1 and PMA1, in a collection of 14 fluconazole-susceptible Candida albicans isolates. For each gene, average expression levels and variations within the population were determined. These values were then used as reference points to make predictions about the molecular basis of resistance in 38 clinical isolates (the majority of which were resistant to fluconazole) obtained from 18 patients treated with posaconazole for refractory oropharyngeal candidiasis. For each of the 38 isolates, the expression levels of genes encoding efflux pumps, ERG11 and the control genes, were measured as above. Comparison of the two data sets revealed that expression of ACT1 and PMA1 did not vary significantly between the two sets of isolates. In contrast, MDR1, ERG11, CDR1, and CDR2 were overexpressed in 3, 4, 14, and 35, respectively, of the isolates from patients treated with azoles. In addition to these changes, the patient isolates all had at least one and often multiple missense mutations in ERG11. Select ERG11 alleles were expressed in Saccharomyces cerevisiae; all of the alleles tested conferred reduced susceptibility to fluconazole. Despite both the increases in pump expression and the ERG11 mutations, only one of the patient isolates exhibited a large decrease in posaconazole susceptibility.
Asunto(s)
Antifúngicos/farmacología , Azoles/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/genética , Actinas/genética , Antifúngicos/uso terapéutico , Candidiasis Bucal/tratamiento farmacológico , Candidiasis Bucal/microbiología , Clonación Molecular , Sistemas de Computación , ADN de Hongos/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Farmacorresistencia Fúngica , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Pruebas de Sensibilidad Microbiana , Mutación/genética , Orofaringe/microbiología , ATPasas de Translocación de Protón/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Saccharomyces cerevisiae/genética , Especificidad de la Especie , Transcripción Genética , Triazoles/farmacología , Triazoles/uso terapéuticoRESUMEN
OBJECTIVES: To characterize the molecular mechanisms responsible for reduced susceptibility to azoles in Candida albicans clinical isolates. MATERIALS AND METHODS: Seven sequential C. albicans isolates were cultured from an AIDS patient treated with posaconazole for refractory oropharyngeal candidiasis. Expression levels of the CDR1, CDR2 and MDR1 genes, encoding efflux pumps previously implicated in azole resistance, and ERG11, encoding the azole target site, were monitored using northern blot and real-time PCR. The ERG11 genes from all seven isolates were sequenced. RESULTS: The seven closely related isolates exhibited significant decreases in susceptibility to fluconazole (MIC >or= 32 mg/L) and voriconazole (MIC >or= 2 mg/L) and progressive decreases in susceptibility to both posaconazole (isolates 1-4 MIC 0.25 mg/L, isolates 5-7 MIC 2 mg/L) and itraconazole (isolates 1-4 MIC 1 mg/L, isolates 5-7 MIC > 8 mg/L). None of the isolates exhibited any significant changes in the expression levels of ERG11 or the efflux pump genes. All seven isolates had multiple mutations in ERG11; isolates one through four each had five missense mutations; four of the resultant amino acid changes were previously associated with azole resistance. The fifth isolate had an additional novel mutation in one copy of ERG11, resulting in a Pro-230 to Leu substitution. This mutation was present in both ERG11 genes in the last two isolates. Select ERG11 genes were expressed in Saccharomyces cerevisiae, the ERG11 allele with all six mutations conferred the highest level of posaconazole resistance. CONCLUSIONS: Multiple mutations in ERG11 are required to confer decreased susceptibility to posaconazole.
Asunto(s)
Antifúngicos/uso terapéutico , Candida albicans/efectos de los fármacos , Candidiasis Bucal/tratamiento farmacológico , Farmacorresistencia Fúngica/genética , Triazoles/uso terapéutico , Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Candida albicans/genética , Candida albicans/aislamiento & purificación , Candidiasis Bucal/microbiología , Genes Virales/genética , Infecciones por VIH/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , MutaciónRESUMEN
To better understand the molecular basis of posaconazole (POS) resistance in Aspergillus fumigatus, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 10(8) and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in A. fumigatus was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (mdr1 and mdr2) and/or alterations in accumulation of [(14)C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of cyp51A or cyp51B, which encode the presumed target site for POS, cytochrome P450 14alpha-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of cyp51A. Mutations at the same locus were identified in three clinical A. fumigatus isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible A. fumigatus strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the cyp51A gene in A. fumigatus can confer reduced susceptibility to POS.