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 rever sibly between two or more general phenotypes, a process now considered a pu tative virulence factor in this species. Candida albicans WO-1 switches fre quently and reversibly between a white and an opaque phase, and this phenot ypic transition is accompanied by the differential expression of white-phas e-specific and opaque-phase-specific genes. In the opaque phase, cells diff erentially 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 c onstructs fused to the Renilla reniformis luciferase was used to functional ly characterize the OP4 promoter in order to investigate how this gene is d ifferentially expressed in the white-opaque transition. An extremely strong 17-bp transcription activation sequence was identified between -422 and -4 04 bp. This sequence contained a MADS box consensus binding site, most clos ely 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. Get mobility shift assays with the 17-bp activation sequence identified thr ee specific complexes which formed with both white- and opaque-phase cell e xtracts. Competition with a putative MADS box consensus binding site from t he promoter of the coordinately regulated opaque-phase-specific gene PEP1 ( SAP1) and the human MADS box consensus binding site for serum response fact or demonstrated that one of the three complexes formed was specific to the OP4 sequence.