Candidosis is one of the most important AIDS- related opportunistic infections, afflicting the majority of AIDS victims. Candida albicans, the primary cause of candidosis, is a member of the normal flora and grows either in yeast of hyphal form. The ability to generate hyphae is pathogenically important. Compared to the yeast form, the hyphal form shows enhanced adherence to oral epithelial cells and enhanced invasion of host tissues. While the mechanisms resulting in the enhanced adherence of hyphal forms are not well understood, it is very likely that hypha-specific surface proteins are involved. Preliminary data presented in this proposal shows that hyphae contain unique surface proteins not found on yeast surfaces. Further, additional preliminary data suggests that a full-length cDNA encoding the major immunodominant hyphal surface protein has been cloned. The hypothesis to be tested is that yeast and hyphal growth forms differentially regulate either the mRNA levels, the translation of mRNA, or the intracellular localization of this hyphal surface protein. The cDNA encoding this protein as well as the recombinant protein produced in vitro, will be used to tesst this hypothesis directly, by identifying mechanisms which control the presence of the mRNA and the protein corresponding to this cDNA. The first specific aim of this proposal is to determine definitively that the cloned cDNA actually encodes a hyphal surface protein, This will be domne by first showing that the recombinant protein and C. albicans hyphal surface protein cross react. In addition, the amino acid sequence predicted by the cDNA insert will be compared to the internal amino acid sequence of peptide fragments of the purified hyphal surface protein. The second and third specific aims are to isolate and sequence a genomic clone encoding the hyphal protein, using a fulll-length cDNA as a probe. Genomic clones will include flanking sequences that potentially contain regulatory elements necessary for gene expression. The fourth specific aim is to initiate studies on mechanisms controlling expression of the unique hyphal surface protein. Preliminary data suggests that this mRNA is present in both yeast and hyphae. This result will be confirmed using slot blot analysis. In addition, mRNA levels will be correlated with hyphal protein levels. The presence in yeast of mRNA encoding a hyphal surface protein is explained by one of two possibilities. Either the mRNA is not translated in yeast, or it is translate is not secreted. Correlation of hyphal protein to mRNA levels will allow these two possibilities to be distinguished. The researdh proposed here is prerequisite for studies of the molecular interactions between C. albicans proteins and host cells. The hypothesis that surface proteins are important in adherence to host cells will be tested by performing in vitro adherence assays, employing the purified protein as an inhibitor. In addition, it will be possible to isolate host cell redeptors to C. albicans via chemical crosslinking. Finally, studies to identify potential targets for anti-fungal therapy will be initiated by mapping adherent as welll as immunodominant domains on the protein.
