DNA repair has not been studied in the human pathogen Candida albicans in spite of numerous observations that suggest extensive genomic variability among clinical isolates. This variability has been associated with a variety of genomic alterations that include, deletions, translocations, ploidy changes, as well as loss of chromosomes or parts of chromosomes. The relationship of these changes to adaptation, virulence, and growth of the fungus in the host is uncertain, but sugar utilization and drug resistance have been correlated in vitro with aneuloploidy. Homologous recombination [HR] and non-homologous end-joining [NHEJ] should be important components in DNA repair to ensure the fidelity of these genomic alterations. Our preliminary studies with Rad52p of the HR and Lig4p of the NHEJ pathways suggest that both proteins provide growth functions in addition to DNA repair in C. albicans. This fact along with the surprising genomic variability of this pathogen suggest differences between the DNA repair pathways of S. cerevisiae and C. albicans. To understand the roles of HR and NHEJ in this organism, we propose four specific aims.
Specific aim 1 includes the construction of new mutants in each of the two pathways, HDF1 in NHEJ and RAD59 in HR.
In specific aim 2, we will complete our analysis of PIF1 and RRM3, two proteins identified from a two-hybrid screen that interact with Lig4p. The role of HR and NHEJ pathway proteins in DNA repair and morphogenesis is the focus of specific aim 3. In addition, microarray analysis will be used to identify up and down regulated genes in selected mutants of C. albicans when compared to wild type cells. Finally, aim 4 is designed to evaluate the role of several gene deleted mutants in the pathogenesis of candidiasis. We propose to use three mouse models of candidiasis to evaluate virulence, including, invasive, oral, and vaginal models so as to identify a site-specific role of these genes in virulence. In summary, our studies will provide critical information on the contribution of DNA repair to the adaptation, growth, variability and virulence of C. albicans.
