Abstract
SummaryDespite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using “air-liquid interface” (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion > 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions.
Highlights
Cryptosporidium is a gastrointestinal parasite that causes longterm illness in immunocompromised patients (O’Connor et al, 2011) and contributes to malnourishment and impaired growth in children in low- to middle-income countries (Kotloff, 2017; Kotloff et al, 2013)
We further demonstrate the utility of this system by generating transgenic parasites and performing an in vitro genetic cross, confirming the Mendelian nature of meiosis and opening forward genetics in C. parvum
MIECs were amplified as spheroids (Miyoshi et al, 2012; Miyoshi and Stappenbeck, 2013; VanDussen et al, 2015), plated onto transwells that contained a matrigel coating and irradiated 3T3 cell feeder layer. mouse intestinal epithelial cells (mIECs) monolayers were first grown submerged in conditioned medium for 7 days, followed by removal of the top medium
Summary
Cryptosporidium is a gastrointestinal parasite that causes longterm illness in immunocompromised patients (O’Connor et al, 2011) and contributes to malnourishment and impaired growth in children in low- to middle-income countries (Kotloff, 2017; Kotloff et al, 2013). Genetic modification of C. parvum using CRISPR/Cas requires propagation in immunocompromised mice (Vinayak et al, 2015), and stable transgenic parasites still cannot be selected or propagated in vitro due to the lack of robust cell culture platforms. Adenocarcinoma cell lines such as HCT-8 and Caco-2 support short-term growth, but the parasite does not complete its life cycle and replication ceases after a few rounds of asexual division (Upton et al, 1994). Because sexual recombination does not readily occur in vitro, little is known about the processes of sexual differentiation, fertilization, or meiosis in this parasite
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