MOLECULAR-CLONING AND CHARACTERIZATION OF A CANDIDA-ALBICANS GENE CODING FOR CYTOCHROME-C HEME LYASE AND A CELL WALL-RELATED PROTEIN

Immunoscreening of a Candida albicans cDNA library with a monoclonal a ntibody (mAb 4C12) recognizing an epitope present in high-molecular-we ight mannoprotein (HMWM) components specific for the mycelial cell wai ls (a 180 kDa component and a polydispersed 260 kDa species) resulted in the isolation of the gene CaCYC3 encoding for cytochrome c haem lya se (CCHL). The CaCYC3 gene was transcribed preferentially in mycelial cells in which two mRNA transcripts of 0.8 and 1 kb were found. The nu cleotide and the deduced amino acid sequences of this gene displayed 4 5% homology and 46% identity, respectively, to the Saccharomyces cerev isiae CYC3 gene and shared common features with other reported genes e ncoding for CCHL. The CaCYC3 gene restored the respiratory activity wh en transformed in a S. cerevisiae cyc3(-) mutant strain. A C. albicans CYC3 null mutant was constructed after sequential disruption using th e hisG::URAS::hisG ('ura-blaster') cassette. Null mutant cells were un able to use lactate as a sole carbon source and had a reduced ability to form germ tubes. Western immunoblotting analysis of subcellular fra ctions from wild-type and null mutant strains demonstrated the presenc e of two gene products, a 33 kDa mitochondrial protein and a 40 kDa ce ll wall-associated moiety reacting with antibodies against CCHL, in bo th yeast cells and germ tubes. mAb 4C12 still reacted with the CaCYC3 null mutant (by immunofluorescence and immunoblotting) but showed an a ltered pattern of immunoreactivity against cell wall HMWM species, ind icating a relationship between these moieties and the CaCYC3 gene prod ucts. The results suggest that the CaCYC3 gene encodes two proteins, o ne targeted to the mitochondria and the other to the cell wall.