Secretion of virulence factors by the fungal pathogen Cryptococcus neoformans

在免疫功能低下的人，如艾滋病毒+/艾滋病患者，病原性的担子菌真菌隐球菌新生经常导致危及生命的脑膜脑炎。此外，还有近缘种C. gattii最近在加拿大的不列颠哥伦比亚省表现为对免疫活性个体的威胁。主要的C新形虫的毒力机制包括多糖荚膜的生物合成、黑色素的产生以及在人类生理温度（37°C）下生长的能力。cAMP依赖性蛋白激酶（PKA）信号通路介导细胞反应，包括营养感测、应激反应、黑色素和荚膜产生以及菌丝生长。C.新变型导致荚膜产生减少和毒力减弱。这种表型在PKA催化亚基缺失的pka 1突变体中观察到。相比之下，pkr 1突变缺乏PKA的调节亚基产生扩大胶囊，是高毒力。 引人注目的是，最近的研究表明，PKA 1和PKR 1突变体改变了分泌途径组分的转录水平。基于这一重要发现，本课题的目的是研究cAMP/PKA通路对C.新人类第一个目标是确定是否外泌和高尔基体-内体途径都影响胶囊的生物合成。因此，外囊蛋白Sec 15和内体蛋白如突触融合蛋白Pep 12的作用将被研究用于囊形成。这些蛋白质的选择是基于PKA调节其转录水平的发现。第二个目标是基于甘露糖蛋白Ova 1受PKA调节并对胶囊大小、黑色素形成和锂敏感性产生负面影响的发现。将进行遗传筛选，以确定可能与Ova 1一起影响囊形成和锂敏感性的其他PKA调节靶蛋白。深入的功能特性，这些目标蛋白质将提高我们的PKA依赖性调节胶囊生产和毒力在这个主要的真菌病原体的机械理解。

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.