A Mutation in Tac1p, a Transcription Factor Regulating CDR1 and CDR2, Is Coupled With Loss of Heterozygosity at Chromosome 5 to Mediate Antifungal Resistance in Candida albicans

Abstract

TAC1, a Candida albicans transcription factor situated near the mating-type locus on chromosome 5, is necessary for the upregulation of the ABC-transporter genes CDR1 and CDR2, which mediate azole resistance. We showed previously the existence of both wild-type and hyperactive TAC1 alleles. Wild-type alleles mediate upregulation of CDR1 and CDR2 upon exposure to inducers such as fluphenazine, while hyperactive alleles result in constitutive high expression of CDR1 and CDR2. Here we recovered TAC1 alleles from two pairs of matched azole-susceptible (DSY294; FH1: heterozygous at mating-type locus) and azole-resistant isolates (DSY296; FH3: homozygous at mating-type locus). Two different TAC1 wild-type alleles were recovered from DSY294 (TAC1-3 and TAC1-4) while a single hyperactive allele (TAC1-5) was isolated from DSY296. A single amino acid (aa) difference between TAC1-4 and TAC1-5 (Asn977 to Asp or N977D) was observed in a region corresponding to the predicted activation domain of Tac1p. Two TAC1 alleles were recovered from FH1 (TAC1-6 and TAC1-7) and a single hyperactive allele (TAC1-7) was recovered from FH3. The N977D change was seen in TAC1-7 in addition to several other aa differences. The importance of N977D in conferring hyperactivity to TAC1 was confirmed by site-directed mutagenesis. Both hyperactive alleles TAC1-5 and TAC1-7 were codominant with wild-type alleles and conferred hyperactive phenotypes only when homozygous. The mechanisms by which hyperactive alleles become homozygous was addressed by comparative genome hybridization and single nucleotide polymorphism arrays and indicated that loss of TAC1 heterozygosity can occur by recombination between portions of chromosome 5 or by chromosome 5 duplication. C ANDIDA albicans is an opportunistic pathogen that causes oral and systemic infections in immunocompromised patients as well as vaginal infections in immunocompetent women. To prevent and treat Candida infections, immunocompromised patients are often treated for a long time with antifungal agents among which is the class of azoles. As azoles are fungistatic, rather than fungicidal, C. albicans cells repetitively exposed to these antifungals can adapt to the drug pressure and eventually become resistant to azoles. The most important mechanism of resistance to azoles is the overexpression of multidrug transporters, encoded by either the major facilitator efflux pump CaMDR1 (multidrug resistance 1) or the ABC transporters CDR1 (candida drug resistance) and CDR2. Upregulation of CaMDR1 confers resistance to fluconazole, while upregulation of CDR1 and CDR2 confers resistance to multiple azoles (itraconazole, fluconazole, voriconazole). Understanding the transcriptional control of these genes, by both cis-and trans-acting effectors, is therefore important for determining how azole resistance and transport mechanisms are regulated in C. albicans.CaMDR1 expression is controlled by at least two regulatory promoter cis-acting regions as reported recently by Harry et al. (2005). Several elements of...

Transcriptional Regulation of MDR1 , Encoding a Drug Efflux Determinant, in Fluconazole-Resistant Candida albicans Strains through an Mcm1p Binding Site

“…2 and Table 4, each strain exhibiting trans-activation of MDR1 or CDR2 had increased levels of MDR1 mRNA or CDR2 mRNA, respectively. Strains exhibiting CDR2 trans-activation also coordinately overexpressed CDR1, as has been previously described for strains that contain hyperactive TAC1 alleles (9). This report focuses on the mechanism(s) whereby MDR1 is trans-activated in FLC R strains; mutations leading to trans-activation of CDR2 will be described in detail elsewhere.…”

“…Sanglard and coworkers identified a transcription factor, Tac1p, that binds an element within the CDR1/CDR2 promoter, the drug resistance element (DRE), and demonstrated its involvement in the high-level constitutive expression of these genes seen in some FLC R strains (10). Mutations within the TAC1 open reading frame (ORF) that lead to hyperactive trans-activation of CDR1 and CDR2 have been identified in FLC R strains (9). Therefore, mutations that result in increased activity of trans-activators occur commonly in FLC R strains.…”

Identification of promoter elements responsible for the regulation of MDR1 from Candida albicans, a major facilitator transporter involved in azole resistance

“…We have shown that Cap1p is likely to be one trans-acting factor for the HRE; however, confirmation of its role and the full identification of the trans-acting components with which it interacts will necessitate further investigation. Future studies will also clarify whether mutations in MCM1 can be responsible for high constitutive expression of MDR1 in a manner analogous to the recent finding that mutations in TAC1, a key trans-acting regulator of the ABC-transporter genes CDR1 and CDR2, are responsible for the high CDR1/ CDR2 expression of clinical strains (Coste et al, 2006).…”

Relative Contributions of the Candida albicans ABC Transporters Cdr1p and Cdr2p to Clinical Azole Resistance

“…It was shown that the upregulation of the CDR1 and CDR2 genes in A r isolates is due to gain-of-function mutations in the zinc cluster transcription factor Tac1p (5,6). Most of these mutations consist of C-terminal amino acid substitutions or small inframe deletions (4,5).…”