Transcriptomic atlas of the morphologic development of the fungal pathogen Coccidioides reveals key phase-enriched transcripts [ChIP-Seq]

Coccidioides spp. are highly understudied but significant dimorphic fungal pathogens that can infect both immunocompetent and immunocompromised people. In the environment, they grow as multicellular filaments (hyphae) that produce vegetative spores called arthroconidia. Upon inhalation by mammals, arthroconidia undergo a process called spherulation. They enlarge and undergo numerous nuclear divisions to form a spherical structure, and then internally segment until the spherule is filled with multiple cells called endospores. Mature spherules rupture and release endospores, each of which can form another spherule, in a process thought to facilitate dissemination. Spherulation is unique to Coccidioides and its molecular determinants remain largely unknown. Here, we report the first high-density transcriptomic analyses of Coccidioides development, defining morphology-dependent transcripts and those whose expression is regulated by Ryp1, a major regulator required for spherulation and virulence. Of approximately 9000 predicted transcripts, we discovered 273 transcripts with consistent spherule-associated expression, 82 of which are RYP1-dependent, a set likely to be critical for Coccidioides virulence. ChIP-Seq revealed 2 distinct regulons of Ryp1, one shared between hyphae and spherules and the other unique to spherules. Spherulation regulation was elaborate, with the majority of 227 predicted transcription factors in Coccidioides displaying spherule-enriched expression. We identified provocative targets, including 20 transcripts whose expression is endospore-enriched and 14 putative secreted effectors whose expression is spherule-enriched, of which 6 are secreted proteases. To highlight the utility of these data, we selected a cluster of RYP1-regulated, arthroconidia-associated transcripts and found that they play a role in arthroconidia cell wall biology, demonstrating the power of this resource in illuminating Coccidioides biology and virulence. Overall design: We generated arthroconidia and grew them as spherules (39°C, 10% CO2) or hyphae (room temp, no additional CO2) in the specified media. At each timepoint, RNA was harvested and the transcriptome was analyzed by RNA-Seq. We have included 3 timecourses of WT spherules, 1 full and 1 limited timecourse of the ryp1? mutant which cannot form the spherule morphology but was subjected to spherulation or hyphal growth conditions. For samples labeled as originating from Figure S4, we also performed ChIP-Seq on the same biological samples using a polyclonal anti-Ryp1 antibody.

球孢子菌属（Coccidioides spp.）是一类研究被极大程度忽视但具有重要致病意义的二态性真菌病原体，可感染免疫功能正常及免疫功能低下人群。在自然环境中，它们以多细胞菌丝（hyphae）形式生长，并产生被称为关节孢子（arthroconidia）的营养孢子。当被哺乳动物吸入后，关节孢子会经历一个被称为球囊形成（spherulation）的发育过程：孢子体积增大并经历多次核分裂，形成球形结构，随后内部发生裂解，直至球囊腔内充满被称为内生孢子（endospores）的子代细胞。成熟球囊破裂并释放内生孢子，每个内生孢子均可再形成一个新的球囊，这一过程被认为有助于病原菌的体内播散。球囊形成是球孢子菌属特有的生物学过程，其分子调控机制在很大程度上仍未明确。
本研究首次开展了球孢子菌属发育进程的高密度转录组分析，鉴定得到形态依赖性转录本，以及受球囊形成与毒力关键调控因子Ryp1调控的转录本。在约9000个预测转录本中，我们筛选出273个与球囊形成稳定相关的转录本，其中82个为Ryp1依赖型转录本，该组转录本可能对球孢子菌属的致病毒力至关重要。
染色质免疫沉淀测序（ChIP-Seq）结果显示，Ryp1存在两类截然不同的调控子：一类在菌丝与球囊中均发挥调控作用，另一类则为球囊特有调控子。球囊形成的调控网络极为复杂，球孢子菌属中预测的227个转录因子中，绝大多数均表现出球囊富集表达特征。本研究还鉴定出多个值得关注的靶标基因，包括20个内生孢子富集表达的转录本，以及14个球囊富集表达的推定分泌效应因子，其中6个为分泌蛋白酶。
为验证本数据集的应用价值，我们选取了一组受Ryp1调控且与关节孢子相关的转录本进行功能验证，发现它们参与关节孢子的细胞壁生物学过程，充分证实了本资源在解析球孢子菌属生物学特性与毒力机制方面的重要作用。
实验设计概况：我们制备了关节孢子，并将其分别在指定培养基中培养为球囊（39℃、10% CO₂）或菌丝（室温、无额外CO₂）。在每个采样时间点收集总RNA，通过RNA测序（RNA-Seq）分析转录组表达谱。本研究包含3组野生型（WT）球囊形成时间进程样本，以及1组完整及1组有限的ryp1缺失突变体（ryp1?）时间进程样本：该突变体无法形成球囊形态，但被置于球囊形成或菌丝生长的培养条件下。对于标记为图S4来源的样本，我们还使用多克隆抗Ryp1抗体对同一批生物学样本开展了ChIP-Seq实验。