Center for Genome Sciences and Systems Biology and Departments of Computer Science and Genetics, Washington University School of Medicine, St. Louis, Missouri, United States of America.
PLoS Pathog. 2011 Dec;7(12):e1002411. doi: 10.1371/journal.ppat.1002411. Epub 2011 Dec 8.
Cryptococcus neoformans is an opportunistic fungal pathogen that causes serious human disease in immunocompromised populations. Its polysaccharide capsule is a key virulence factor which is regulated in response to growth conditions, becoming enlarged in the context of infection. We used microarray analysis of cells stimulated to form capsule over a range of growth conditions to identify a transcriptional signature associated with capsule enlargement. The signature contains 880 genes, is enriched for genes encoding known capsule regulators, and includes many uncharacterized sequences. One uncharacterized sequence encodes a novel regulator of capsule and of fungal virulence. This factor is a homolog of the yeast protein Ada2, a member of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex that regulates transcription of stress response genes via histone acetylation. Consistent with this homology, the C. neoformans null mutant exhibits reduced histone H3 lysine 9 acetylation. It is also defective in response to a variety of stress conditions, demonstrating phenotypes that overlap with, but are not identical to, those of other fungi with altered SAGA complexes. The mutant also exhibits significant defects in sexual development and virulence. To establish the role of Ada2 in the broader network of capsule regulation we performed RNA-Seq on strains lacking either Ada2 or one of two other capsule regulators: Cir1 and Nrg1. Analysis of the results suggested that Ada2 functions downstream of both Cir1 and Nrg1 via components of the high osmolarity glycerol (HOG) pathway. To identify direct targets of Ada2, we performed ChIP-Seq analysis of histone acetylation in the Ada2 null mutant. These studies supported the role of Ada2 in the direct regulation of capsule and mating responses and suggested that it may also play a direct role in regulating capsule-independent antiphagocytic virulence factors. These results validate our experimental approach to dissecting capsule regulation and provide multiple targets for future investigation.
新生隐球菌是一种机会性真菌病原体，在免疫功能低下的人群中会引起严重的人类疾病。其多糖荚膜是一个关键的毒力因子，可响应生长条件而调节，在感染的情况下会增大。我们使用微阵列分析了在一系列生长条件下刺激形成荚膜的细胞，以鉴定与荚膜增大相关的转录特征。该特征包含 880 个基因，富含编码已知荚膜调节剂的基因，并且包含许多未被描述的序列。一个未被描述的序列编码一种新型荚膜和真菌毒力的调节剂。该因子是酵母蛋白 Ada2 的同源物，是 Spt-Ada-Gcn5 乙酰转移酶（SAGA）复合物的成员，通过组蛋白乙酰化调节应激反应基因的转录。与这种同源性一致，C. neoformans 缺失突变体表现出组蛋白 H3 赖氨酸 9 乙酰化减少。它还对各种应激条件表现出缺陷，表现出与其他具有改变的 SAGA 复合物的真菌重叠但不相同的表型。该突变体在有性发育和毒力方面也存在显著缺陷。为了在荚膜调节的更广泛网络中确定 Ada2 的作用，我们对缺失 Ada2 或两种其他荚膜调节剂（Cir1 和 Nrg1）之一的菌株进行了 RNA-Seq 分析。结果分析表明，Ada2 通过高渗甘油（HOG）途径的成分在 Cir1 和 Nrg1 下游发挥作用。为了鉴定 Ada2 的直接靶标，我们对 Ada2 缺失突变体中的组蛋白乙酰化进行了 ChIP-Seq 分析。这些研究支持了 Ada2 在荚膜和交配反应的直接调节中的作用，并表明它可能在调节非荚膜相关抗吞噬毒力因子方面也发挥直接作用。这些结果验证了我们用于剖析荚膜调节的实验方法，并为未来的研究提供了多个目标。
