title: Disruption of the transcriptional regulator Cas5 results in enhanced killing of Candida albicans by Fluconazole.
abstract: Azole antifungal agents such as fluconazole exhibit fungistatic activity against Candida albicans. Strategies to enhance azole antifungal activity would be therapeutically appealing. In an effort to identify transcriptional pathways that influence the killing activity of fluconazole, we sought to identify transcription factors (TFs) involved in this process. From a collection of C. albicans strains disrupted for genes encoding TFs (O. R. Homann, J. Dea, S. M. Noble, and A. D. Johnson, PLoS Genet. 5:e1000783, 2009, http://dx.doi.org/10.1371/journal.pgen.1000783), four strains exhibited marked reductions in minimum fungicidal concentration (MFCs) in both RPMI and yeast extract-peptone-dextrose (YPD) media. One of these genes, UPC2, was previously characterized with regard to its role in azole susceptibility. Of mutants representing the three remaining TF genes of interest, one (CAS5) was unable to recover from fluconazole exposure at concentrations as low as 2 μg/ml after 72 h in YPD medium. This mutant also showed reduced susceptibility and a clear zone of inhibition by Etest, was unable to grow on solid medium containing 10 μg/ml fluconazole, and exhibited increased susceptibility by time-kill analysis. CAS5 disruption in highly azole-resistant clinical isolates exhibiting multiple resistance mechanisms did not alter susceptibility. However, CAS5 disruption in strains with specific resistance mutations resulted in moderate reductions in MICs and MFCs. Genome-wide transcriptional analysis was performed in the presence of fluconazole and was consistent with the suggested role of CAS5 in cell wall organization while also suggesting a role in iron transport and homeostasis. These findings suggest that Cas5 regulates a transcriptional network that influences the response of C. albicans to fluconazole. Further delineation of this transcriptional network may identify targets for potential cotherapeutic strategies to enhance the activity of the azole class of antifungals.
authors.affiliation: Department of Clinical Pharmacy, College of Pharmacy, Memphis, Tennessee, USA.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Vasicek
authors.affiliation: Department of Clinical Pharmacy, College of Pharmacy, Memphis, Tennessee, USA.
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Berkow
authors.affiliation: Department of Microbiology and Immunology, Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA.
authors.forename: Vincent M
authors.initials: VM
authors.lastname: Bruno
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.
authors.forename: Aaron P
authors.initials: AP
authors.lastname: Mitchell
authors.affiliation: School of Medicine, University of Texas Health Science Center, San Antonio, Texas, USA.
authors.forename: Nathan P
authors.initials: NP
authors.lastname: Wiederhold
authors.affiliation: Department of Clinical Pharmacy, College of Pharmacy, Memphis, Tennessee, USA.
authors.forename: Katherine S
authors.initials: KS
authors.lastname: Barker
authors.affiliation: Department of Clinical Pharmacy, College of Pharmacy, Memphis, Tennessee, USA Department of Pediatrics, College of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, USA Children's Foundation Research Center at Le Bonheur Children's Medical Center, Memphis, Tennessee, USA progers3@uthsc.edu.
authors.forename: P David
authors.initials: PD
authors.lastname: Rogers
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects*; D002177:Candidiasis / Q000188:drug therapy / Q000382:microbiology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics; D015725:Fluconazole / Q000494:pharmacology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D055786:Gene Knockout Techniques; D053263:Gene Regulatory Networks / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000935:Antifungal Agents; D002352:Carrier Proteins; C511850:Cas5 protein, Candida albicans; D014157:Transcription Factors; D015725:Fluconazole; D003577:Cytochrome P-450 Enzyme System