Cultivation of host-adaptedCryptosporidium parvumandCryptosporidium hominisusing enteroids for cryopreservation of isolates and transcriptomic studies of infection

Abstract

ABSTRACTCryptosporidium hominisandCryptosporidium parvumare major causes of severe diarrhea in humans. Comparative studies of them are hampered by the lack of effective cultivation and cryopreservation methods, especially forC. hominis. Here, we described adapted murine enteroids for the cultivation of oneC. parvumIId subtype and nonhuman primate-adaptedC. hominisIb, Im, and In subtypes, which allowed the complete development of the pathogens, producing oocysts infectious to mice. Using the system, we developed a novel cryopreservation method forCryptosporidiumisolates. In comparative RNA-seq analyses ofC. hominiscultures, the enteroid system generated significantly more transcriptomic responses of both pathogen and host genes than the conventional HCT-8 cell system. In particular, the infection was shown to upregulate PI3K-Akt, Wnt, Ras,TNF, NF-κB, IL-17, MAPK, and innate immunity signaling pathways and downregulate Wnt and Hippo signaling pathways, host cell metabolism, and parasites in enteroid cultures had significantly higher expression of genes involved in oocyst formation. Therefore, the new culture model provides a valuable tool for comparative studies of the biology of divergentCryptosporidiumspecies.IMPORTANCEThe two dominant species for human cryptosporidiosis,Cryptosporidium hominisandCryptosporidium parvum, differ significantly in host range and virulence. Up to date, biological studies ofCryptosporidiumspp. are almost exclusively done with bovine-adapted IIa subtypes ofC. parvum, which is the species with effective laboratory animal models and in vitro cultivation methods. Here, we describe modified procedures for the generation of murine enteroids for successful cultivation of both nonhuman primate-adaptedC. hominissubtypes and aC. parvumIId subtype, producing oocysts infective to mice. In addition, we have developed a novel cryopreservation method using the system for long-term storage ofCryptosporidiumisolates. RNA-seq analyses ofC. hominiscultures indicate that the enteroid culture system generates host and pathogen transcriptomic responses similar to those in natural infection. This new development alleviates a technical bottleneck in cryptosporidiosis research, and provides an example for other difficult-to-culture pathogens of major public health importance.