Pheromones are hormone-like peptides secreted into the growth medium by cells. The binding of each pheromone to it cognate receptor triggers the mating response within the cell. In S. cerevisiae, the response includes induction of a specific set of genes, cell cycle arrest specifically in the G1 stage, and a change in cell morphology. Therefore, pheromones can potentially be used to control growth of yeast cells. Cryptococcus neoformans exists as a yeast like that of S. cerevisiae, and when appropriate strains of C. neoformans ar co-cultured, they are capable of mating to form basidia,, which give rise to meiotic products. This process resembles mating of S. cerevisiae. It is likely that these two yeasts share other elements of a mating program, i.e., they both utilize specific pheromones to cause cell cycle arrest and initiate the mating program. If cryptococcal cells do, indeed, use pheromones to initiate the mating program, these pheromones have promise as a novel and highly specific means to control the growth of C. neoformans. We propose to demonstrate the existence of pheromones in C. neoformans and to examine their ability to control cell cycle progression by pursuit of five specific aims: First, the growth-arresting effects of pheromones of cryptococcal cells will be characterized. Specific issues to be addressed are i) characterization of growth arresting effects by pheromones, ii) effects of pheromones on cell viability, and iii) effects of pheromones on various aspects of yeast physiology. second, pheromones produced by C. neoformans will be isolated and characterized. The peptide amino acid sequence will be determined as well as any modifications of the peptides. Third, C. neoformans pheromone genes will be cloned and expressed in S. cerevisiae. This cloning will facilitate identification of the peptide portion and provide an alternative source of pheromones. Fourth, methods for large-scale production of cryptococcal pheromones will be developed. Approaches to be used are direct isolation from C. neoformans cultures, direct isolation from S. cerevisiae cultures expressing the cryptococcal pheromone genes, and chemical synthesis based on the physical analysis of the pheromone and the deduced amino acid sequence from the DNA sequence. Finally, the in vivo properties of pheromones and liposome-encapsulated pheromones will be evaluated. Properties to be examined are i) serum half life, ii) toxicity, and iii) ability to alter the course of experimental cryptococcosis.
Showing the most recent 10 out of 29 publications
