How polarity is established and maintained during Candida albicans hyphal growth remains largely unknown and this gap in knowledge has limited the development of models for how tissue invasion occurs and how it can be prevented. The long-term goal associated with this research program is to understand how polarity is established in hyphae and how polarity establishment proteins influence C. albicans morphogenesis and invasion properties. The objective of this application is to determine how a comically-localized GTPase (Rsr1 p) controls polarity establishment specifically at hyphal tips. Rsr1 p is required for guiding the direction of hyphal growth, for the ability to invade into agar, and for the localization of cytoskeletal targets of the major polarity establishment GTPase Cdc42p. The central hypothesis of this application is that the cortical GTPase activity of Rsrlp controls the axis of polarity establishment and, hence, the direction of hyphal growth by regulating the localized activation of Cdc42p, and/or its effector(s), at hyphal tips. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Identify the domains of Rsrlp that are required for its function in polarity establishment in hyphae; 2) Determine the effector(s) of Rsrlp activity; and 3) Identify novel C. albicans genes that are important for polarity establishment in hyphae. Under the first aim, versions of Rsrlp lacking predicted functional domains, constructed by PCR-mediated gene modification and site-directed mutagenesis, will be analyzed for defects in polarity establishment, GTPase activity, and localization. Under the second aim, genetic interactions between RSR1 and CDC42 will be determined by a) analyzing double mutant strains for synthetic lethality and b) over-expression of RSR1 for suppression of cdc42 phenotypes. In addition, localization of Cdc42p and its effectors will be determined in strains lacking Rsr1 p activity, using GFP fusion technology. Under the third aim, a published set of novel C. albicans ORFs (that are important for polarized growth, and that lack orthologs in other organisms) will be analyzed for cortical localization of the expressed proteins using GFP fusions. Strains lacking the identified cortically-localized proteins will be constructed and analyzed for defects in polarity establishment during hyphal growth. The proposed research is significant because it will identify putative molecular targets for the development of anti-fungal strategies that prevent tissue invasion. At the same time, the fundamental new knowledge obtained about polarity establishment in hyphae is expected to advance the general field of polarity responses in other filamentous fungi and in other polarized eukaryotic cell systems. This is an important and under-investigated area of C. albicans biology that has applicability to understanding the pathogenesis of tissue invasion during systemic C, albicans infections in immunocompromised patients. ? ? ?
