Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

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-T) 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.

CANDIDA 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 CaMDRl (multirfrug resistance 1) or the ABC transporters CDRl (candida rfrug resistance) and CDR2. Upregulation of CaMDRl confers resistance to fluconazole, while upregulation of CDRl and CDR2 confers resistance to multiple azoles (itraconazole, fluconazole, voriconazole). Understanding the transcriptional control of these genes, by both cis- and irons-acting effectors, is therefore important for determining how azole resistance and transport mechanisms are regulated in C. albicans.

CaMDRl expression is controlled by at least two regulatory promoter as-acting regions as reported recently by HARRY et al. (2005). Several elements of CDR genes are important for the regulation of CDRl and CDR2. A fasal response dement (BRE) is located between nt -860 and -810 in the CDRl promoter, and a rfrug response dement (DRE) is present in the promoters of both CDRl and CDR2 (DE MICHELI et al. 2002). The BRE regulates basal expression of CDRl (DE MICHELI et al. 2002), while the DRE sequence (5'CGGAA/TATCGGATA-3') is crucial for the upregulation of these genes in azole-resistant strains as well as for the transient upregulation of both genes in the presence of different drugs such as oestradiol, progesterone, or fluphenazine in azole-susceptible strains. In addition, another BRE (located between -243 and -234) and a negative regulatory dement (NRE) located within the -289 region have been reported in CDRl (PuRi et al. 1999; GAUR et al. 2004). Finally, in the same gene, KARNANI et al. (2004) identified SREl and SRE2 (steroid response elements) between -696 and -521. …

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