Non-cyp51A-mutation Mediated Triazole Resistance in Aspergillus fumigatus

A critical barrier to overcoming triazole resistance in Aspergillus fumigatus is the significant lack ofunderstanding of its genetic and molecular basis. We have shown that the known mechanisms of resistance donot fully explain resistance observed among most clinical isolates. Our long-term goal is to improve antifungaltherapy and ensure the sustained clinical utility of the triazole class for treatment of infections caused byAspergillus species. Our central hypothesis is that non-cyp51A-mutation mediated mechanisms are essentialto triazole resistance in clinical isolates of A. fumigatus and involve complex genetic changes altering 1) sterolbiosynthesis and its transcriptional activation, 2) triazole transport and its transcriptional activation, and 3) as yetunknown mechanisms. Our current objective is to address critical knowledge gaps by identifying the geneticand molecular determinants of non-cyp51A-mutation mediated resistance. Our preliminary data suggest thatwhile mutations in cyp51A among triazole resistant clinical isolates are common, their overall contribution toresistance is minimal. We have observed mutations, unique to resistant isolates in our collection, in genesencoding sterol sensing proteins, regulators of sterol biosynthesis, and sterol biosynthesis enzymes. We havealso observed clinical isolates that overexpress not only cyp51A, but most genes of the ergosterol biosynthesispathway, suggesting its constitutive activation. We have observed several potential transporters that are up-regulated among triazole resistant isolates in our collection, suggesting a role for triazole efflux and resistanceby these transporters. We have also shown that clinical isolates of A. fumigatus take up triazole antifungals viafacilitated diffusion and we believe that altered triazole import may represent an important mechanism ofresistance. To accomplish our objective we will undertake experiments that will lead to an understanding of whatgenetic and molecular determinants influence triazole susceptibility through altered sterol biosynthesis or itstranscriptional activation (Aim 1) and triazole transport and its regulation (Aim 2). In Aim 3, we will also utilize anunbiased whole genome comparisons, coupled with in vitro evolution experiments, to identify completely novelmechanisms of resistance in clinical isolates. Our approach is innovative as we will use the latest genetic andgenomic techniques to study and discover novel non-cyp51A-mutation mediated mechanisms of triazoleresistance that are operative in a U.S.-based collection of triazole resistant clinical isolates. The proposedresearch is significant as it represents a comprehensive analysis of the molecular and genetic basis of non-cyp51A-mutation mediated triazole resistance in A. fumigatus and will provide novel insights into ways in whichtriazole activity can be improved against this important human pathogen.