MOLECULAR MARKER FOR INVASIVE CANDIDA ALBICANS

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The opportunistic pathogen Candida albicans adheres to epithelium and replicates at the mucosal barrier prior to invading the bloodstream. We have recently characterized an "integrin-like protein" that is antigenically, structurally, and functionally related to the alpha subunits of the beta2 integrins alphaM (Mac-1 or CD11b/CD18) and alphaX (p150,95 or CD11c/CD18)--two members of the leukocyte adhesion glycoprotein family. A major function of this protein is the facilitation of epithelial and endothelial adhesion by recognition of RGD sequences in proteins such as iC3b and fibrinogen that serve as substrates for adhesion. In attainment of the specific aims of the previous five-year funding period, we have now cloned a gene (alpha INT1) encoding a novel surface protein in C. albicans and have analyzed its structure. Like the alpha-subunits of the beta2 integrins, the alpha INT1 gene product contains a ligand binding site for iC3b and fibrinogen, two EF-hand divalent cation binding sites, a hydrophobic transmembrane domain, and a tyrosine residue in the cytoplasmic tail. Alpha INT1 also encodes its own RGD tripeptide. Transformation of S. cerevisiae with the coding sequence of alpha INT1 under the control of a galactose-inducible promoter induces germ tube formation in haploid yeast. In Specific Aim One, a series of deletion mutants lacking the ligand-binding site, one or both divalent cation binding sites, the RGD sequence, or the transmembrane domain an/or cytoplasmic tail will be expressed in E. coli or S. cerevisiae and analyzed for functional deficits in six assays: a) adhesion to epithelial monolayers; b) iC3b-binding ELISA; c) agglutination; d) formation of germ tubes in S. cerevisiae; and e/f) binding of monoclonal and polyclonal antibodies. Field emission scanning electron microscopy will be utilized to confirm surface expression in C. albicans blastospores and to localize intracellular reservoirs for deletion mutants that are not surface-borne. In Specific Aim Two, those domains of alpha INt1p that mimic fibrinogen-binding sites in prokaryotes will be mapped wih the use of deletion mutants, and the relevant regions of fibrinogen (Fbg) will be defined with purified Fbg, isolated gamma-chain, D/E fragments, and internal peptides of Fbg. In specific Aim Three, heterozygous and homozygous null mutants in hand will be used to study the role of alpha INT1 in adhesion and morphogenesis in C. albicans. In Specific Aim Four, we will test the possible interaction of alpha Int1p with the G alpha subunit and 3 cytoskeletal proteins from S. cerevisiae, and with human beta-subunits using gene knockouts, the yeast two-hybrid system, and CHO cell transfection. The studies outlined above should provide novel information regarding the role of alpha Int1p in the processes of adhesion, invasion, and morphogenesis in C. albicans and thereby bring forward new strategies for the control of candidal infection.