Secretion of virulence factors by the fungal pathogen Cryptococcus neoformans

在免疫功能低下的人群中，如HIV+/艾滋病患者，致病性担子菌真菌新隐球菌经常引起危及生命的脑膜脑炎。此外，近缘种加蒂弓形虫最近在加拿大不列颠哥伦比亚省表现出对免疫能力强的个体的威胁。主要的新生芽孢杆菌毒力机制包括多糖胶囊的生物合成、黑色素的产生以及在人体生理温度（37℃）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，包括营养感知、应激反应、黑色素和荚膜生成以及菌丝生长。在新生C.中降低PKA活性导致荚膜产量减少和毒力减弱。这种表型在缺乏PKA催化亚基的pka1突变体中观察到。相反，缺乏PKA调控亚基的pkr1突变体产生增大的荚膜，具有高毒力。引人注目的是，最近的研究表明，pka1和pkr1突变体改变了分泌途径成分的转录水平。基于这一重要发现，本项目的目的是研究cAMP/PKA通路对新生C.囊出口分泌机制的影响。第一个目的是确定胞外途径和高尔基-内体途径是否都影响胶囊的生物合成。因此，将研究外囊蛋白Sec15和内体蛋白（如syntaxin Pep12）在胶囊形成中的作用。这些蛋白的选择是基于PKA调节其转录水平的发现。第二个目标是基于甘露蛋白Ova1受PKA调控，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Ova1一起影响胶囊形成和锂敏感性的pka调控靶蛋白。深入了解这些靶蛋白的功能特征将增强我们对这种主要真菌病原体中pka依赖性胶囊生产和毒力调节的机制理解。

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.