Control of Aspergillus fumigatus polarized growth through regulation of Ras prote

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DESCRIPTION (provided by applicant): I am currently a senior post-doctoral fellow transitioning to an independent investigator. My immediate objective is to complete my current research projects in the Steinbach lab that will lay the foundation for my transition to an independent academic position. My long term career objectives are to establish an internationally-recognized laboratory investigating fungi that cause human disease, with an emphasis on filamentous fungal pathogens. The K22 career award will be particularly important for establishing a vibrant, extramurally funded research laboratory. Aspergillus fumigatus is a commonly isolated, opportunistic mould. The conidia of A. fumigatus are inhaled and undergo germination, giving way to sustained polarized growth. This highly polarized tip growth allows A. fumigatus to invade lung tissues. Despite current antifungal therapy, invasive aspergillosis (IA) caused by A. fumigatus continues to have high morbidity and mortality in immunocompromised patients. Despite the essential nature of polarized growth to the development of IA, little is known about the molecular mechanisms of polarized growth in A. fumigatus. Previously, I identified that the A. fumigatus RasA protein is necessary for proper polarized growth and hyphal morphogenesis. This proposal will test two main hypotheses: (1) Plasma membrane localization of RasA activity, through palmitoylation of the RasA protein, is required for polarized growth and, therefore, virulence in A. fumigatus, and (2) a fungal-specific RasA domain, identified in my preliminary data, is required for RasA GTPase levels needed to support sustained polarized growth. In Aim 1, palmitoylation-mediated regulation of RasA activity will be analyzed by determining the morphological effects of palmitoylation deficient RasA proteins. To define the importance of plasma membrane localization, strains expressing mutant rasA alleles lacking appropriate palmitoylation motifs will be examined for RasA localization, hyphal morphogenesis throughout development, and virulence. In Aim 2, I will analyze the importance of a novel, fungal-specific protein domain to regulation of RasA GTPase activity. In addition, the role of the fungal-specific RasA domain in interaction with regulatory proteins will be explored using in vitro binding assays. Completion of this aim will define the contribution of the fungal-specific Ras domain to A. fumigatus growth and virulence. Upon completion of this proposal, I will have defined the importance of Ras protein compartmentalization to hyphal morphogenesis and virulence and will also have identified regulatory mechanisms of this process. This will enable me to develop more detailed models of differential Ras protein function at specific sub-cellular sites, describing how compartmentalization of Ras activity modulates polarized growth. In addition, I will define the importance of a novel, fungal-specific protein domain to RasA function. Due to the essential nature of Ras proteins to fungal morphogenesis, the identification and characterization of fungal-specific properties of Ras proteins carries the potential of uncovering novel anti-fungal therapies.