PROJECT SUMMARY Candida albicans is a normal commensal of the human microbiota that is also capable of causing superficial and disseminated infections in at-risk patient populations. Immunocompromised individuals and those with head and neck cancers are highly susceptible to Candida infections, which frequently manifest as oropharyngeal candidiasis. These infections are typically caused by the formation of a biofilm, a resistant, surface-adhered microbial community that develops on mucosal surfaces of the mouth or on dental implants within the oral cavity. Formation of a biofilm provides C. albicans with unique properties, such as increased resistance to antifungal treatments and the host immune response. It is not uncommon for cells in a biofilm to be several orders of magnitude more resistant to an antimicrobial agent compared to free-floating cells of the same species. After treatment of a biofilm-based infection in the oral cavity, persistent surviving cells can cause treatment failures and permit reinfection of the area. We hypothesize that the antifungal resistance of C. albicans in the biofilm state is regulated by a complex transcriptional network of “master” transcriptional regulators and their downstream targets. This proposal aims to discover the complete transcriptional network controlling resistance mechanisms in C. albicans biofilms. In Aim 1, we screen a comprehensive library of homozygous transcription factor (TF) deletion mutants using in vitro biofilm assays to identify regulators with altered growth in the presence of antifungals commonly prescribed to treat oral C. albicans infections. In Aim 2, we validate our in vitro findings using an in vivo rat biofilm model of denture stomatitis. In Aim 3, we map the connections of each transcription factor to one another as well as to downstream target genes to determine the regulatory network controlling resistance in C. albicans biofilms. This work will significantly expand our understanding of antifungal resistance mechanisms and may lead to the development of novel therapeutics against biofilm infections.