Department of Medicine, University of Vermont Larner College of Medicine, Burlington, Vermont, United States of America.
Cell, Molecular, and Biomedical Sciences Graduate Program, University of Vermont, Burlington, Vermont, United States of America.
PLoS Pathog. 2024 Apr 26;20(4):e1011906. doi: 10.1371/journal.ppat.1011906. eCollection 2024 Apr.
The apicomplexan parasite Cryptosporidium is a leading cause of childhood diarrhea in developing countries. Current treatment options are inadequate and multiple preclinical compounds are being actively pursued as potential drugs for cryptosporidiosis. Unlike most apicomplexans, Cryptosporidium spp. sequentially replicate asexually and then sexually within a single host to complete their lifecycles. Anti-cryptosporidial compounds are generally identified or tested through in vitro phenotypic assays that only assess the asexual stages. Therefore, compounds that specifically target the sexual stages remain unexplored. In this study, we leveraged the ReFRAME drug repurposing library against a newly devised multi-readout imaging assay to identify small-molecule compounds that modulate macrogamont differentiation and maturation. RNA-seq studies confirmed selective modulation of macrogamont differentiation for 10 identified compounds (9 inhibitors and 1 accelerator). The collective transcriptomic profiles of these compounds indicates that translational repression accompanies Cryptosporidium sexual differentiation, which we validated experimentally. Additionally, cross comparison of the RNA-seq data with promoter sequence analysis for stage-specific genes converged on a key role for an Apetala 2 (AP2) transcription factor (cgd2_3490) in differentiation into macrogamonts. Finally, drug annotation for the ReFRAME hits indicates that an elevated supply of energy equivalence in the host cell is critical for macrogamont formation.
顶复亚门寄生虫隐孢子虫是发展中国家儿童腹泻的主要病因。目前的治疗选择并不充分，多种临床前化合物正被积极探索作为隐孢子虫病的潜在药物。与大多数顶复门生物不同，隐孢子虫属在同一宿主内顺序进行无性繁殖，然后进行有性繁殖，以完成其生命周期。抗隐孢子虫化合物通常通过仅评估无性阶段的体外表型测定来鉴定或测试。因此，专门针对有性阶段的化合物仍未被探索。在这项研究中，我们利用 ReFRAME 药物再利用文库针对新设计的多读数成像测定法来识别调节大配子体分化和成熟的小分子化合物。RNA-seq 研究证实了 10 种鉴定化合物（9 种抑制剂和 1 种促进剂）对大配子体分化的选择性调节。这些化合物的集体转录组谱表明，伴随着隐孢子虫有性分化，翻译抑制伴随发生，我们通过实验验证了这一点。此外，对 RNA-seq 数据与阶段特异性基因启动子序列分析的交叉比较表明，AP2 转录因子（cgd2_3490）在分化为大配子体中起着关键作用。最后，ReFRAME 命中的药物注释表明，宿主细胞中能量等价物的供应增加对于大配子体形成至关重要。
