Mendelian segregation and high recombination rates facilitate genetic analyses inCryptosporidium parvum

Abstract

ABSTRACTVery little is known about the process of meiosis in the apicomplexan parasiteCryptosporidiumdespite the essentiality of sex in its life cycle. Most cell lines only support asexual growth ofCryptosporidium parvum(C. parvum), but stem cell derived intestinal epithelial cells grown under air-liquid interface (ALI) conditions support the sexual cycle. To examine chromosomal dynamics during meiosis inC. parvum, we generated two transgenic lines of parasites that were fluorescently tagged with mCherry or GFP on chromosomes 1 or 5, respectively. Infection of ALI cultures orIfngr1-/-mice with mCherry and GFP parasites produced “yellow” oocysts generated by cross-fertilization. Outcrossed oocysts from the F1generation were purified and used to infect HCT-8 cultures, and phenotypes of the progeny were observed by microscopy. All possible phenotypes predicted by independent segregation were represented equally (∼25%) in the population, indicating thatC. parvumchromosomes exhibit a Mendelian inheritance pattern. Unexpectedly, the most common pattern observed from the outgrowth of single oocysts included all possible parental and recombinant phenotypes derived from a single meiotic event, suggesting a high rate of crossover. To estimate the frequency of crossover, additional loci on chromosomes 1 and 5 were tagged and used to monitor intrachromosomal crosses inIfngr1−/−mice. Both chromosomes showed a high frequency of crossover compared to other apicomplexans with map distances (i.e., 1% recombination) of 3-12 kb. Overall, a high recombination rate may explain many unique characteristics observed inCryptosporidiumspp. such as high rates of speciation, wide variation in host range, and rapid evolution of host-specific virulence factors.AUTHOR SUMMARYAlthough sex is essential for the transmission and maintenance of infection ofCryptosporidium, it has been historically challenging to study the process of meiosis in this medically relevant protist. We utilize recent methodological advances such as a specializedin vitroculture system, cell sorting, and the generation of transgenic parasites to cross identical strains of parasites in the absence of selection pressure to identify intrinsic chromosome behavior during meiosis. By specifically examining the phenotypes from the first generation of parasites, we reveal that cross-fertilization frequently occurs in parasite populations, chromosomes segregate in a Mendelian manner, and the rate of crossover is high on Chromosomes 1 and 5. Understanding these baseline meiotic mechanisms is essential for planning and interpreting future genetic studies ofCryptosporidiumseeking to identify genes associated with phenotypes of interest.