Department of Microbiology, University of Georgia, Athens, GA 30602, USA.
Genetics. 2024 Jul 8;227(3). doi: 10.1093/genetics/iyae059.
Cryptococcus neoformans is a fungal pathogen of the top critical priority recognized by the World Health Organization. This clinically important fungus also serves as a eukaryotic model organism. A variety of resources have been generated to facilitate investigation of the C. neoformans species complex, including congenic pairs, well-annotated genomes, genetic editing tools, and gene deletion sets. Here, we generated a set of strains with all major organelles fluorescently marked. We tested short organelle-specific targeting sequences and successfully labeled the following organelles by fusing the targeting sequences with a fluorescence protein: the plasma membrane, the nucleus, the peroxisome, and the mitochondrion. We used native cryptococcal Golgi and late endosomal proteins fused with a fluorescent protein to label these two organelles. These fluorescence markers were verified via colocalization using organelle-specific dyes. All the constructs for the fluorescent protein tags were integrated in an intergenic safe haven region. These organelle-marked strains were examined for growth and various phenotypes. We demonstrated that these tagged strains could be employed to track cryptococcal interaction with the host in phagocytosis assays. These strains also allowed us to discover remarkable differences in the dynamics of proteins targeted to different organelles during sexual reproduction. Additionally, we revealed that "dormant" spores transcribed and synthesized their own proteins and trafficked the proteins to the appropriate subcellular compartments, demonstrating that spores are metabolically active. We anticipate that these newly generated fluorescent markers will greatly facilitate further investigation of cryptococcal biology and pathogenesis.
新生隐球菌是世界卫生组织认定的具有最高优先级别（top critical priority）的真菌病原体。这种具有临床重要意义的真菌也是一种真核模式生物。为了促进对新生隐球菌种复合体的研究，已经产生了多种资源，包括同基因对、注释良好的基因组、遗传编辑工具和基因缺失集。在这里，我们生成了一套主要细胞器均被荧光标记的菌株。我们测试了短的细胞器特异性靶向序列，并通过将靶向序列与荧光蛋白融合，成功标记了以下细胞器：质膜、核、过氧化物酶体和线粒体。我们使用与荧光蛋白融合的天然隐球菌高尔基体和晚期内体蛋白来标记这两个细胞器。通过细胞器特异性染料的共定位来验证这些荧光标记。所有荧光蛋白标签的构建体都整合在一个基因间安全港区域。对这些细胞器标记的菌株进行了生长和各种表型的检测。我们证明这些标记菌株可用于跟踪隐球菌在吞噬作用测定中与宿主的相互作用。这些菌株还使我们能够发现蛋白质在有性生殖过程中靶向不同细胞器的动态存在显著差异。此外，我们揭示了“休眠”孢子转录和合成自身的蛋白质，并将蛋白质运送到适当的亚细胞隔室，表明孢子具有代谢活性。我们预计这些新生成的荧光标记将极大地促进对隐球菌生物学和发病机制的进一步研究。
