Table_4_Autophagy Regulates Fungal Virulence and Sexual Reproduction in Cryptococcus neoformans.DOCX

Autophagy (macroautophagy) is an evolutionarily conserved degradation pathway involved in bulk degradation of cytoplasmic organelles, old protein, and other macromolecules and nutrient recycling during starvation. Extensive studies on functions of autophagy-related genes have revealed that autophagy plays a role in cell differentiation and pathogenesis of pathogenic fungi. In this study, we identified and characterized 14 core autophagy machinery genes (ATGs) in C. neoformans. To understand the function of autophagy in virulence and fungal development in C. neoformans, we knocked out the 14 ATGs in both α and a mating type strain backgrounds in C. neoformans, respectively, by using biolistic transformation and in vivo homologous recombination. Fungal virulence assay showed that virulence of each atgΔ mutants was attenuated in a murine inhalation systemic-infection model, although virulence factor production was not dramatically impaired in vitro. Fungal mating assays showed that all the 14 ATGs are essential for fungal sexual reproduction as basidiospore production was blocked in bilateral mating between each atgΔ mutants. Fungal nuclei development assay showed that nuclei in the bilateral mating of each atgΔ mutants failed to undergo meiosis after fusion, indicating autophagy is essential for regulating meiosis during mating. Overall, our study showed that autophagy is essential for fungal virulence and sexual reproduction in C. neoformans, which likely represents a conserved novel virulence and sexual reproduction control mechanism that involves the autophagy-mediated proteolysis pathway.

自噬（巨自噬，macroautophagy）是一类进化保守的降解途径，参与饥饿状态下细胞质细胞器、衰老蛋白质及其他大分子的批量降解与营养循环。针对自噬相关基因功能的大量研究表明，自噬参与病原真菌的细胞分化与致病过程。本研究在新型隐球菌（C. neoformans）中鉴定并表征了14个核心自噬机制相关基因（autophagy-related genes，ATGs）。为阐明自噬在新型隐球菌毒力与真菌发育中的功能，我们分别在α和a两种交配型的新型隐球菌菌株背景中，通过基因枪转化法与体内同源重组技术，敲除了这14个ATGs。真菌毒力测定结果显示，在小鼠吸入性全身感染模型中，各atgΔ突变株的毒力均出现显著衰减，尽管体外培养时其毒力因子的产生未受到明显损害。真菌交配实验表明，14个ATGs均为真菌有性生殖所必需：各atgΔ突变株的双侧交配过程中，担孢子的产生被完全阻断。细胞核发育实验显示，各atgΔ突变株的双侧交配融合后，细胞核无法完成减数分裂，这表明自噬对于调控交配过程中的减数分裂至关重要。综上，本研究证实自噬对新型隐球菌的毒力与有性生殖均不可或缺，这可能代表了一种由自噬介导的蛋白水解途径参与的保守新型毒力与有性生殖调控机制。