Transcriptional regulation of MDR1, encoding a drug efflux determinant, in fluconazole-resistant Candida albicans strains through an Mcm1p binding site.

Candida albicans is a commensal organism of the human gastrointestinal tract and genitourinary tract but can become an important opportunistic pathogen, especially in immunocompromised individuals (32). Depending on the underlying immune defect, C. albicans causes a variety of disease states, including superficial mucosal infections and more serious disseminated infections that exhibit approximately 40% attributable mortality (24). C. albicans has been associated with the AIDS epidemic from the outset (21). Oropharyngeal candidiasis, or thrush, in seemingly healthy individuals is often the first indication of human immunodeficiency virus (HIV) infection, and most AIDS patients have one or more episodes of Candida infection (22, 52). Fluconazole is the drug of choice for treating candidal infection in AIDS patients due to its low toxicity and oral bioavailability (7, 18, 53). However, the widespread use of fluconazole to treat candidal infections has led to the emergence of resistant Candida strains, and resistance is now a clinical problem (22, 51, 52). In one study, approximately one-third of late-stage AIDS patients carried drug-resistant C. albicans in their oral cavities (35). Fluconazole-resistant (FLCR) C. albicans is now seen in both HIV and non-HIV patients who have never been exposed to fluconazole (38). Commonly seen molecular mechanisms for fluconazole resistance in C. albicans include increased efflux of fluconazole and mutation of ERG11, the gene encoding the target of fluconazole, lanosterol demethylase. Mutations within the ERG11 structural gene reduce fluconazole binding (41, 53). The most common mechanism, occurring in approximately 85% of clinical isolates, is increased efflux mediated by overexpression of the genes encoding the multidrug resistance efflux proteins Mdr1p, Cdr1p, and Cdr2p (41). Mdr1p is classified in the major facilitator superfamily of efflux transporters and exports a variety of structurally unrelated compounds, including fluconazole, benomyl, methotrexate, cycloheximide, 4-nitroquinoline-N-oxide, and sulfometuron methyl (6). C. albicans cells lacking the MDR1 gene are hypersensitive to most of the drugs listed above (20). In two FLCR clinical isolates, deletion of MDR1 abolished the drug resistance phenotype of these strains, demonstrating that in these strains, fluconazole resistance was dependent on the overexpression of MDR1 (55). Moreover, engineered overexpression of MDR1 confers increased resistance to some compounds (28). Despite the frequency with which MDR1-overexpressing, FLCR strains are isolated, the molecular mechanisms leading to high-level expression of MDR1 are not well understood (37). In some FLCR strains, high-level expression of MDR1 is due to the effects of an undefined trans-acting factor(s) (54). Recent work by Hiller et al. indicates that three independent regions of the MDR1 promoter are capable of contributing to MDR1 expression in a FLCR strain overexpressing MDR1 and that the portion of the MDR1 promoter they term region 2 (−588 to −500) mediates benomyl induction of MDR1 transcription in the FLC-sensitive (FLCS) laboratory strain CAI4 (29). In another study, analyzing the FLCS C. albicans laboratory strain CAI8, Harry et al. identified two or more cis-acting promoter regions that contribute to MDR1 expression (25). Under the conditions used by Harry et al., the more-proximal cis-acting element (−399 to −299) was responsible for benomyl-induced transcription of MDR1 while the more-distal cis-acting element (−601 to −500) was implicated in MDR1 induction by oxidizing agents. The most-distal region identified by both groups (−601/−588 to −500) contains a sequence that resembles and functions like the YAP1-responsive element of Saccharomyces cerevisiae (25). Yap1p is a member of the bZIP family of transcription factors and regulates the S. cerevisiae FLR1 gene, encoding a multidrug efflux protein that is similar to C. albicans Mdr1p (1). The homologous C. albicans gene is termed CAP1 (for C. albicans AP-1) (2). Surprisingly, deletion of CAP1 in C. albicans did not lead to FLC hypersensitivity (2). When the CAP1 gene was disrupted in a laboratory-derived FLCR strain that overexpresses MDR1, increased levels of MDR1 mRNA were observed, indicating that Cap1p acts as a negative regulator of MDR1 in this strain and is not responsible for the preexisting MDR1 overexpression. Therefore, other uncharacterized protein factors probably bind to elements within the MDR1 promoter and trans-activate the MDR1 gene in FLCR strains. Expression of the other drug efflux transporters, Cdr1p and Cdr2p, is also regulated through a trans-activating factor that binds a specific sequence within their promoters (10, 11). Cdr1p and Cdr2p are ATP binding cassette (ABC) transporters that are highly similar to each other in sequence (46). Increased expression of these genes has been observed in FLCR clinical strains, and deletion of the CDR1 gene in C. albicans results in hypersensitivity to the azole drugs fluconazole, ketoconazole, and itraconazole and to other drugs such as terbinafine and amorolfine (47). The CDR2 gene also confers resistance to the azoles, terbinafine, and amorolfine (46). 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 FLCR strains (10). Mutations within the TAC1 open reading frame (ORF) that lead to hyperactive trans-activation of CDR1 and CDR2 have been identified in FLCR strains (9). Therefore, mutations that result in increased activity of trans-activators occur commonly in FLCR strains. In this study, we demonstrate that a promoter element termed the MDRE (for “MDR1 drug resistance element”) mediates MDR1 trans-activation in five independent FLCR, MDR1-overexpressing strains, indicating a common mechanism for trans-activation. The MDRE acts in a dosage-dependent and orientation-independent manner to increase transcription of MDR1 in drug-resistant, trans-activation strains. The protein Mcm1p binds the MDRE, and mutations within the MDRE that abolish Mcm1p binding abolish MDR1 trans-activation. Extracts from MDR1 trans-activation strains contain higher levels of MDRE binding activity. The sequence of MCM1 is wild type in these FLCR strains, indicating that the activity rather than the structure of Mcm1p is altered in the trans-activating mutants. Therefore, these results demonstrate that Mcm1p participates in the regulation of expression from the MDR1 promoter at the MDRE.