Table_3_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）是一种在进化过程中得以保留的降解途径，参与细胞质细胞器、老旧蛋白以及其他大分子和营养物质的降解，并在饥饿状态下进行营养物质的循环利用。对自噬相关基因功能的广泛研究揭示了自噬在细胞分化和病原真菌致病机理中的重要作用。在本研究中，我们鉴定并表征了新型隐球菌（C. neoformans）中的14个核心自噬机制基因（ATGs）。为探究自噬在新型隐球菌的致病性和真菌发育中的作用，我们分别利用生物力转化和体内同源重组技术，在α和a交配型菌株背景下敲除了这14个ATGs。真菌致病性实验表明，在鼠吸入性系统性感染模型中，每个atgΔ突变体的致病性均有所降低，尽管在体外其致病因子产生并未受到显著损害。真菌交配实验显示，所有14个ATGs对于真菌的有性繁殖至关重要，因为在每个atgΔ突变体之间的双向交配中，担孢子产生被阻断。真菌核发育实验表明，在双向交配的每个atgΔ突变体中，融合后的核未能进行减数分裂，这表明自噬对于调节交配过程中的减数分裂是必不可少的。总体而言，我们的研究显示自噬对于新型隐球菌的致病性和有性繁殖至关重要，这很可能代表了一种保守的新型致病性和有性繁殖调控机制，该机制涉及自噬介导的蛋白质降解途径。