Korwin-Mihavics Bethany R, Dews Emmett A, Miller Peter, Cameron Alexandra, Martorelli di Genova Bruno, Huston Christopher D
Department of Medicine, University of Vermont, Burlington, Vermont, USA.
Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, USA.
Microbiol Spectr. 2025 Jun 25:e0050225. doi: 10.1128/spectrum.00502-25.
Many advances have been made recently in our understanding of 's asexual cycle and sexual differentiation. However, the process of fertilization, which is required for transmission of infectious oocysts, is not well understood. Typical cancer cell-based culture only allows robust exploration of asexual cycle and sexual differentiation of . To facilitate exploration of sexual reproduction in we developed an organoid-based culture system that supports full life cycle and a novel fertilization reporter. Organoid-derived monolayers (ODMs) supported fertilization and oocyst production and maintained the infection for up to 3 weeks. ODM-derived oocysts were infectious . Fertilization was confirmed by successfully mating two strains of and with a novel fertilization switch reporter. The fertilization switch reporter utilizes a DiCre system in which cre fragments are expressed under the control of sexual stage promoters, resulting in a rapamycin-inducible switch in fluorescent protein expression from mCherry to mNeonGreen after fertilization that is spatially and temporally controlled. This results in mCherry-positive parasites in the first generation and offspring that express mNeonGreen. validation of the fertilization switch reporter demonstrated the precision and efficiency of the fertilization switch reporter and confirmed excision of the mCherry gene sequence only after rapamycin treatment. The start of a second generation of parasites was also shown in the ODMs and rarely in HCT8s. Use of this reporter in ODMs can help investigate the life cycle post-sexual differentiation in a physiologically relevant system.IMPORTANCEOrganoid-derived monolayers enable the study of previously inaccessible aspects of 's biology. This system overcomes the disadvantages of previous organoid-based methods for culture. It is faster and simpler than previously described systems, uses defined differentiation media to increase reproducibility, enables real-time observation, supports parasite fertilization and oocyst production, and provides a physiologically relevant tissue culture system to facilitate cell biology studies. The organoid-derived monolayer system could facilitate the study of host-pathogen interactions, -host specificity, or innate or cellular immune responses to infection stimulated in the intestinal epithelium. The fertilization switch reporter could be used to test factors or drugs that may have potential to interfere with sexual reproduction. Organoid-based cell cultures in combination with the fertilization switch reporter could increase our understanding of sexual reproduction in leading to vital information for the development of sexual reproduction inhibitors or vaccines that could shorten disease duration and prevent transmission.
最近，我们对[寄生虫名称]的无性繁殖周期和有性分化的理解有了许多进展。然而，对于感染性卵囊传播所必需的受精过程，我们还了解得不够透彻。典型的基于癌细胞的培养仅能有力地探索[寄生虫名称]的无性繁殖周期和有性分化。为了便于探索[寄生虫名称]的有性生殖，我们开发了一种基于类器官的培养系统，该系统支持[寄生虫名称]的完整生命周期，并带有一种新型受精报告基因。类器官衍生的单层细胞（ODMs）支持受精和卵囊产生，并能维持感染长达3周。ODMs衍生的卵囊具有感染性。通过成功使两株[寄生虫名称]交配，并使用新型受精开关报告基因，证实了受精过程。该受精开关报告基因利用DiCre系统，其中cre片段在性阶段启动子的控制下表达，导致受精后荧光蛋白表达从mCherry到mNeonGreen的雷帕霉素诱导开关，且具有时空控制。这导致第一代寄生虫和后代中mCherry阳性寄生虫表达mNeonGreen。对受精开关报告基因的验证证明了受精开关报告基因的精确性和效率，并证实仅在雷帕霉素处理后mCherry基因序列被切除。在ODMs中也显示出了第二代寄生虫的开始，而在HCT8s中很少见。在ODMs中使用该报告基因有助于在生理相关的[寄生虫名称]系统中研究有性分化后的[寄生虫名称]生命周期。
类器官衍生的单层细胞能够研究[寄生虫名称]生物学中以前难以触及的方面。该系统克服了以前基于类器官的[寄生虫名称]培养方法的缺点。它比先前描述的系统更快、更简单，使用确定的分化培养基来提高可重复性，能够进行实时观察，支持寄生虫受精和卵囊产生，并提供一个生理相关的组织培养系统以促进细胞生物学研究。类器官衍生的单层细胞系统可以促进对宿主 - 病原体相互作用、[寄生虫名称] - 宿主特异性或肠道上皮中对[寄生虫名称]感染刺激的先天或细胞免疫反应的研究。受精开关报告基因可用于测试可能干扰[寄生虫名称]有性生殖的因素或药物。基于类器官的细胞培养与受精开关报告基因相结合，可以增加我们对[寄生虫名称]有性生殖的理解，从而为开发可缩短疾病持续时间并防止传播的有性生殖抑制剂或疫苗提供重要信息。
