Organoid-based in vitro system and reporter for the study of Cryptosporidium parvum sexual reproduction

ABSTRACT Many advances have been made recently in our understanding of Cryptosporidium’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 Cryptosporidium. To facilitate exploration of sexual reproduction in Cryptosporidium parvum, we developed an organoid-based culture system that supports Cryptosporidium’s 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 in vivo. Fertilization was confirmed by successfully mating two strains of C. parvum 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. In vivo 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 Cryptosporidium life cycle post-sexual differentiation in a physiologically relevant in vitro system.IMPORTANCEOrganoid-derived monolayers enable the study of previously inaccessible aspects of Cryptosporidium’s biology. This system overcomes the disadvantages of previous organoid-based methods for Cryptosporidium 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, Cryptosporidium-host specificity, or innate or cellular immune responses to Cryptosporidium 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 Cryptosporidium’s sexual reproduction. Organoid-based cell cultures in combination with the fertilization switch reporter could increase our understanding of sexual reproduction in Cryptosporidium, leading to vital information for the development of sexual reproduction inhibitors or vaccines that could shorten disease duration and prevent transmission.