Effects of Gain-of-Function Mutations in Pdr1 on Fluconazole Resistance and Global Gene Expression in Candida glabrata

K. E. CAUDLE1, N. P. WIEDERHOLD 2, K. S. BARKER 1, P. D. ROGERS 1;

Author address: 

1Univ. of TN, Memphis, TN, 2Univ. of TX, San Antonio, TX.

Abstract: 

Background: Azole antifungal resistance in Candida glabrata is primarily mediated by the zinc cluster transcription factor Pdr1p. Mutations in PDR1 result in constitutive up-regulation of Pdr1p target genes. We used matched azole susceptible and resistant isolates to delineate the Pdr1p regulon and to determine if different mutations in PDR1 result in different levels of azole resistance and different target gene activation. Methods: Five azole susceptible and resistant matched isolate pairs were used in this study. PDR1 alleles were sequenced to identify differences between matched susceptible and resistant isolates. PDR1 alleles with putative gain-of-function mutations were expressed in both a common background and parent strain in which PDR1 had been disrupted. Fluconazole susceptibilities were determined by CLSI microdilution. Expression of PDR1 targets were measured by real-time RT-PCR in these strains. Genome-wide gene expression profiles were determined by microarray analysis. Results: Two putative novel gain-of-function mutations were identified in two of the matched isolates. One resistant isolate had no PDR1 mutation. Introduction of the hyperactive alleles into a common strain disrupted for PDR1 conferred different levels of resistance. Likewise, introduction of these alleles into their respective parent isolates resulted in increased expression of Pdr1p targets, but to varying degrees. Microarray analysis comparing these strains to their respective parent identified a core set of commonly differentially expressed genes as well as genes uniquely regulated by specific mutations. Conclusions: These studies have defined a core set of genes regulated by Pdr1p and demonstrate that different mutations in PDR1 have different effects on both fluconazole susceptibility and target gene expression. Moreover, these results suggest the existence of a mechanism for constitutive activation of the Pdr1p regulon independent of mutations in PDR1.
2009

abstract No: 

M-1926

Full conference title: 

49th Annual ICAAC
    • ICAAC 49th