Watson Lucy C, Sala Katarzyna A, Bernitz Netanya, Baumgärtel Lotta, Pallett Mitchell A, Marzook N Bishara, Straker Lorian Cobra, Peng Duo, Collinson Lucy, Sateriale Adam
Cryptosporidiosis Laboratory, The Francis Crick Institute, London, UK.
Electron Microscopy Science Technology Platform, The Francis Crick Institute, London, UK.
Nat Commun. 2025 Aug 20;16(1):7749. doi: 10.1038/s41467-025-63012-1.
The Cryptosporidium parasite is one of the leading causes of diarrheal morbidity and mortality in children, and adolescent infections are associated with chronic malnutrition. There are no vaccines available for protection and only one drug approved for treatment that has limited efficacy. A major barrier to developing new therapeutics is a lack of foundational knowledge of Cryptosporidium biology, including which parasite genes are essential for survival and virulence. Here, we iteratively improve the tools for genetically manipulating Cryptosporidium and develop a targeted CRISPR-based screening method to rapidly assess how the loss of individual parasite genes influence survival in vivo. Using this method, we examine the parasite's pyrimidine salvage pathway and a set of leading Cryptosporidium vaccine candidates. From this latter group, using inducible knockout, we determined the parasite gene known as Cp23 to be essential for survival in vivo. Parasites deficient in Cp23 were able to replicate within and emerge from infected epithelial cells, yet unable to initiate gliding motility which is required for the reinfection of neighbouring cells. The targeted screening method presented here is highly versatile and will enable researchers to more rapidly expand the knowledge base for Cryptosporidium infection biology, paving the way for new therapeutics.
隐孢子虫是导致儿童腹泻发病和死亡的主要原因之一，青少年感染与慢性营养不良有关。目前尚无用于预防的疫苗，仅有一种获批用于治疗的药物，其疗效有限。开发新疗法的一个主要障碍是缺乏对隐孢子虫生物学的基础知识，包括哪些寄生虫基因对生存和毒力至关重要。在此，我们反复改进用于基因操作隐孢子虫的工具，并开发了一种基于CRISPR的靶向筛选方法，以快速评估单个寄生虫基因的缺失如何影响其在体内的生存。使用这种方法，我们研究了寄生虫的嘧啶补救途径以及一组主要的隐孢子虫疫苗候选物。从后一组中，通过诱导敲除，我们确定了名为Cp23的寄生虫基因对体内生存至关重要。缺乏Cp23的寄生虫能够在受感染的上皮细胞内复制并从中出现，但无法启动相邻细胞再感染所需的滑行运动。本文介绍的靶向筛选方法具有高度通用性，将使研究人员能够更快地扩展隐孢子虫感染生物学的知识库，为新疗法铺平道路。
