This proposal describes our ongoing efforts to characterize the eight ALS (agglutinin-like sequence) genes of Candida albicans and determine the function of their encoded proteins. In the previous funding period, we generated a set of alsA/alsA mutant strains and evaluated their phenotype in assays for adhesion to host cells and molecules, and for their ability to cause damage in disease models. Data showed that several of the Als proteins contribute to adhesion of C. albicans to host surfaces and suggested other intriguing roles for the Als proteins. This competing continuation application includes experiments that utilize well-characterized Als proteins to define adhesive function at the molecular level and also pursues investigations to define the multifunctional nature of the Als family.
In Aim 1, the strong adhesive function of AlsSp is exploited to isolate the host cell proteins to which it binds;experiments to demonstrate the specificity of the interaction are also described. Surface plasmon resonance is utilized to further characterize these molecular interactions and random mutagenesis is used to define amino acids required for adhesive function. The focus of Aim 2 is ranking Als2p within the Als family for its relative contribution to adhesion to host cells and also to further define the role of Als2p and Alslp in regulation of C. albicans cell size.
Aim 3 expands on previous experiments that showed that mutagenesis of ALS5, ALS6 or ALS7 resulted in increased adhesion of C. albicans to host surfaces. Previous microarray analysis showed that deletion of these genes results in up-regulation of other C. albicans cell wall proteins that might have adhesive function.
Aim 4 makes use of our large collection of ALS-related strains and constructs to produce Als protein-specific monoclonal antibodies. These antibodies will be used to define Als protein localization on the surface of various C. albicans morphological forms, during cell mating, and on fungal cells from various animal disease models. We will also use the antibodies to support our conclusions about the role of Als proteins in C. albicans adhesion to host surfaces. Data from these studies are key to understanding Als adhesive interactions at the molecular level and understanding the breadth of function within the Als family.
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