ERG11-Mediated Azole Resistance in Candida albicans

Candida species are commensal to the gut or colonizers of the skin, these organisms have the propensity to cause disease. The emergence of antifungal resistant clinical isolates creates problems in antifungal prophylaxis and empirical treatment strategies. The azole-antifungal class has been antifungal pharmacotherapy for 20 years. As the only oral antifungal treatment, the azoles are the most suitable treatment required for antifungal prophylaxis and therapy.;Candida albicans is the cause of fungal disease. Overexpression of the efflux transporter genes CDR1, CDR2, and MDR1 is a mechanism of drug resistance in C. albicans and the majority of investigations defining mechanisms of transcriptional regulation of efflux transporters. Point mutations in the ERG11 gene, whose gene product is the target of azoles, result in reduced target binding affinity. In addition to point mutations, overexpression of ERG11 has also been shown to decrease fluconazole susceptibility. ERG11 gene amplification by chromosome 5 duplication or the presence of a chr5L isochromosome is known to contribute to azole resistance. The zinc-cluster transcription factor Upc2 has been shown to regulate the expression of ERG11.;In a group of clinical C. albicans isolates, I created a transcriptional profile defining expression of genes known to cause azole resistance such as ERG11, CDR1, CDR2 and MDR1. CDR1 and CDR2 overexpression was coordinately regulated and quite prevalent among these isolates. Of those isolates that did overexpress MDR1, even fewer isolates expressed MDR1 to the levels previously observed in azole-resistant isolates. ERG11 was found to be upregulated in almost three-fourths of the fluconazole-resistant isolates. This suggests that ERG11 overexpression is a common contributor to fluconazole resistance in C. albicans. Among the ERG11-overexpressing isolates, I recovered eight distinct single-nucleotide substitutions in UPC2. Five of these substitutions in UPC2 have not been described previously. Four mutations resulted in increased ERG11 expression and increased resistance to fluconazole but to various degrees. Genome-wide transcriptional analysis was performed vi for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by the MDR1 gene, and the uncharacterized ATP binding cassette transporter CDR11. These findings demonstrate that gain-of-function mutations in UPC2 are prevalent among clinical isolates, and make a significant contribution to azole antifungal resistance, but the findings do not account for ERG11 overexpression in all such isolates of C. albicans.;Not all ERG11-overexpression of isolates could be explained by GOF mutations in Upc2. In C. albicans, the Pho-G transcription factor NDT80 has been implicated in azole resistance not only due to its regulation of CDR1 but also due to its regulation of genes involved in the ergosterol biosynthesis pathway. NDT80 alleles for genetically matched pairs of isolates 945/1619 and 1002/3795 were sequenced. In both matched sets, the fluconazole resistant isolate overexpresses ERG11. Sequencing of the NDT80 allele of both matched sets revealed several mutations that resulted in amino acid substitutions when compared to SC5314. This analysis also showed that a loss of heterozygostiy event occurs so that the resistant counterpart was homozygous for one allele. A strain carrying the NDT80 allele derived from fluconazole-resistant isolate 1619 did not result in increased ERG11 expression and increased fluconazole resistance. The mechanism by which ERG11 is upregulated in the absence of UPC2 gain-of-function mutations is currently under investigation.;In addition to ERG11-overexpression, mutations in ERG11 that result in amino acid substitutions in lanosterol demethylase associated with decreased azole susceptibility. I examined ERG11 mutations in the same group clinical C. albicans isolates. I identified that 55 of the 63 isolated contained missense mutations in ERG11 that resulted in at least one amino acid substitution. A selected a group of mutant ERG11 alleles was expressed in an azole-susceptible background so I could determine the specific contribution of the mutant ERG11 allele on antifungal susceptibility. I was interested in characterizing amino acid substitutions. In total, I characterized ten ERG11 alleles containing one amino acid substitution and nine alleles which carried ERG11 alleles with amino acid substitutions. Fluconazole, itraconazole and voriconazole susceptibilities for these strains were tested. Many of these mutations resulted in fluconazole resistance, but most were not significant when tested against voriconazole or itraconazole. Itraconazole, in particular seemed less effected by ERG11 mutations which produced significant resistance to fluconazole although amino acid combination Y132F and F145L resulted in increased itraconazole resistance. Specific combinations of ERG11 mutations resulted in increased azole resistance beyond single mutations. These data suggest that structural differences between azole effect activity against specific mutant ERG11 alleles.