A new pathway for azole resistance in Aspergillus fumigatus



 DESCRIPTION (provided by applicant): Invasive Aspergillosis caused by azole resistant A. fumigatus has an alarming 12% survivability making this a clinical problem of acute significance. Early work on azole resistant isolates of A. fumigatus suggested that azole resistance was a relatively rare occurrence and that the genetic basis of resistance was most often due to changes in a gene (cyp51A) encoding the azole target protein, lanosterol ¿-14 demethylase. Extensive studies from a group in the Netherlands provided compelling evidence that in that country, most azole resistant A. fumigatus isolates contained a single compound mutation consisting of a duplicated region of the cyp51A promoter region (TR34) and a change in the coding sequence altering the amino acid sequence of the protein (L98H). However, more recent studies from a group in the United Kingdom indicated that alterations in the cyp51A gene were unlikely to explain all azole resistance in A. fumigatus. More than 50% of azole resistant isolates from UK patients have wild-type versions of cyp51A, inconsistent with changes at this gene explaining drug tolerance. In 2013, the UK group identified an ATP-binding cassette (ABC) transporter-encoding gene called cdr1B as being associated with azole resistance in several patient isolates. Expression of cdr1B was elevated in highly azole tolerant clinical isolates while the cyp51A remained wild-type in sequence. Overexpression of ABC transporter-encoding genes resulting in azole tolerance has been the predominant route of azole resistance in the Candida species and we believe this will emerge as a key feature of drug resistance in A. fumigatus. This application will focus on the contributions of the cdr1B gene as expression of this ABC transporter is elevated in azole resistance isolates and loss of cdr1B produces an azole sensitive phenotype. We will directly compare the azole resistance phenotypes of wild-type and cdr1B¿ cells containing various cyp51A mutations that are associated with clinically relevant drug resistance. We will also evaluate expression of cyp51A using immunological probes to determine if these mutant alleles influence protein levels; information that is currently unavailable in A. fumigatus. We will carry out a forward genetic screen using impala transposon mutagenesis to identify genes that modulate cdr1B expression. Together, these approaches will provide important new information into the molecular basis of azole resistance in A. fumigatus and provide the direct assessment of drug resistance contributions from ABC transporters and the cyp51A gene.