Secretion of virulence factors by the fungal pathogen Cryptococcus neoformans

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In immunocompromised persons such as HIV+/AIDS-patients, the pathogenic basidiomycetous fungus Cryptococcus neoformans frequently causes life-threatening meningoencephalitis. In addition, the related species C. gattii has recently manifested itself as a threat to immunocompetent individuals in British Columbia, Canada. The major C. neoformans virulence mechanisms are comprised of the biosynthesis of a polysaccharide capsule, the production of melanin, and the ability to grow at the physiological temperature of humans (37°C). The cAMP-dependent protein kinase (PKA) signalling pathway mediates cellular responses including nutrient sensing, stress responses, melanin and capsule production, and hyphal growth. Compromising PKA activity in C. neoformans results in decreased capsule production and attenuated virulence. This phenotype is obesrved in a pka1 mutant which lacks the catalytic subunit of PKA. In contrast, a pkr1 mutant lacking the regulatory subunit of PKA produces an enlarges capsule and is hypervirulent. Strikingly, recent investigations have shown that the pka1 and pkr1 mutants have altered transcript levels for secretory pathway components. Based on this important discovery, the aim of this project is to investigate the influence of the cAMP/PKA pathway on the secretory machinery for capsule export in C. neoformans. The first objective is to determine whether the exocytic and Golgi-endosomal pathways both influence capsule biosynthesis. Therefore, the roles of the exocyst protein Sec15, and endosomal proteins such as the syntaxin Pep12, will be investigated for capsule formation. These proteins have been chosen based on the finding that PKA regulates their transcript levels. The second objective is based on the discovery that the mannoprotein Ova1 is regulated by PKA and negatively affects capsule size, melanin formation and lithium sensitivity. Genetic screens will be performed in order to identify additional PKA-regulated target proteins that may function with Ova1 to influence capsule formation and lithium sensitivity. An in depth functional characterization of these target proteins will enhance our mechanistic understanding of PKA-dependent regulation of capsule production and virulence in this major fungal pathogen.