Our goal is to develop a simple, systematic protein-protein interaction assay in Candida albicans. This organism is the major invasive fungal pathogen of humans, and has been the focus of increasingly mechanistic studies, driven by advances in molecular and genomic technologies and resources. There is growing evidence that numerous unique regulatory pathways govern vital aspects of C. albicans growth and pathogenicity. Protein-protein interaction studies have been critical for pathway analysis in other organisms, but the relevant methodology for C. albicans has lagged behind other developments. We have developed a new protein-protein interaction test, the Vesicle Capture Interaction (VCI) assay that employs highly conserved eukaryotic machinery and minimal genetic manipulation. Interaction is detected through directed localization of fluorescent fusion proteins to endosome surfaces, yielding a punctate fluorescent signal. Images are evaluated through computational methods in order to limit observer bias in interpretation. The VCI assay detects interactions between two protein pairs in both the model yeast Saccharomyces cerevisiae and in C. albicans. We propose to develop a broadly applicable C. albicans VCI assay platform, including strains, vectors, analytical software, and a basic understanding of the gene expression parameters that govern detection. VCI analysis of C. albicans regulatory pathways will open new avenues toward development of therapeutics and a mechanistic understanding of pathogenicity. Conservation of assay components in all known eukaryote suggests that the VCI assay may be imported into other eukaryotic pathogens as well. PUBLIC HEALTH RELEVANCE: Candida albicans is the major invasive fungal pathogen of humans, causing lethal deep tissue infections and severe mucosal infections. This project will implement a new method to identify C. albicans genes that govern infection and drug sensitivity. The outcome will expand the ways in which new antifungal drugs can be developed and tested.