A MADS box protein consensus binding site is necessary and sufficient for activation of the opaque-phase-specific gene OP4 of Candida albicans

The majority of strains of Candida albicans can switch frequently and reversibly between two or more general phenotypes, a process now considered a putative virulence factor in this species. Candida albicans WO-1 switches frequently and reversibly between a white and an opaque phase, and this phenotypic transition is accompanied by the differential expression of white-phase-specific and opaque-phase-specific genes. In the opaque phase, cells differentially express the gene OP4, which encodes a putative protein 402 amino acids in length that contains a highly hydrophobic amino-terminal sequence and a carboxy-terminal sequence with a pI of 10.73. A series of deletion constructs fused to the Renilla reniformis luciferase was used to functionally characterize the OP4 promoter in order to investigate how this gene is differentially expressed in the white-opaque transition. An extremely strong 17-bp transcription activation sequence was identified between -422 and -404 bp. This sequence contained a MADS box consensus binding site, most closely related to the Mcm1 binding site of Saccharomyces cerevisiae. A number of point mutations generated in the MADS box consensus binding site as well as a complete deletion of the consensus site further demonstrated that it was essential for the activation of OP4 transcription in the opaque phase. Gel mobility shift assays with the 17-bp activation sequence identified three specific complexes which formed with both white- and opaque-phase cell extracts. Competition with a putative MADS box consensus binding site from the promoter of the coordinately regulated opaque-phase-specific gene PEP1 (SAP1) and the human MADS box consensus binding site for serum response factor demonstrated that one of the three complexes formed was specific to the OP4 sequence.