Molecular & cellular basis for host-pathogen interactions

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EXCEED THE SPACE PROVIDED. Aspergillus fumigatus (Af) is the major cause of invasive aspergillosis (IA), an often-fatal disease in the growing population of immunocompromised individuals. Itraconazole and amphotericin B are the only currently available treatments for those cases where invasive hyphal growth escapes the host's innate immune system. However, their effectiveness in vivo is low and the prognosis for individuals with IA is poor. The search for (At) virulence factors has been problematic because mutations in the obvious candidates do not reduce virulence. Interestingly, the majority of genes that affect pathogenesis slow the rate of hyphal extension, perhaps giving the host more time to mount a defense. Af grows as a mycelium that extends through medium via polarized hyphal growth. This mode of growth requires that new materials be constantly moved to the growing tip of the hypha. Our laboratory has been involved in elucidating the function of DopA, the founding member of a protein family that is evolutionary conserved fromfungi to mammals. Earlier work from our lab indicates that DopA is an essential gene that is involved in protein trafficking. Specific Aim 1 will test the hypothesis that the ability to establish and maintain highly polarized hyphal growth is a key mechanism for avoidance of innate immunity in immunocompromised patients, and is a major contributing factor in virulence of filamentous fungal pathogens. Mutations that affect fungal growth will be studied in animal cell lines. The virulence of dopA mutants will be assessed in the mouse invasive pulmonary aspergillosis model. Specific Aim 2 will test the hypothesis that Af DopA (DopeyA) and An DopA proteins represent a novel and essential component of cellular protein trafficking required for polarized cell growth in response to environmental signaling. DopA protein will be localized in living cells by using GFP fusions. The role of DopA in the localization of other known polarity determinants will be investigated. DopA interacting proteins will be identified by a number of methods to elucidate the mechanisms of polar growth. This approach will contribute to an understanding of the molecular mechanisms that link signaling to hyphal growth. Specific Aim 3 will test the hypothesis that Af has a cryptic sexual cycle that can be manipulated to develop meiotic genetics as a tool for Af. The addition of sexual genetics will be invaluable to investigators studying Aspergillus fumigatus.