Abstract
The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrhoeal disease and an important contributor to early childhood mortality. Currently, there are no fully effective treatments or vaccines available. Parasite transmission occurs through ingestion of oocysts, through either direct contact or consumption of contaminated water or food. Oocysts are meiotic spores and the product of parasite sex. Cryptosporidium has a single-host life cycle in which both asexual and sexual processes occur in the intestine of infected hosts. Here, we genetically engineered strains of Cryptosporidium to make life cycle progression and parasite sex tractable. We derive reporter strains to follow parasite development in culture and in infected mice and define the genes that orchestrate sex and oocyst formation through mRNA sequencing of sorted cells. After 2 d, parasites in cell culture show pronounced sexualization, but productive fertilization does not occur and infection falters. By contrast, in infected mice, male gametes successfully fertilize female parasites, which leads to meiotic division and sporulation. To rigorously test for fertilization, we devised a two-component genetic-crossing assay using a reporter that is activated by Cre recombinase. Our findings suggest obligate developmental progression towards sex in Cryptosporidium, which has important implications for the treatment and prevention of the infection.
Highlights
The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrhoeal disease and an important contributor to early childhood mortality
Immortalized epithelial cell lines such as Caco[2], HT-29 and HCT-8 are readily infected, but growth ceases after 3 d and the infection cannot be maintained by serial passage[11] (Fig. 1b)
We introduced a fusion of Cryptosporidium parvum histone H2B with the fluorescent reporter mNeon[16] (Supplementary Fig. 1)
Summary
The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrhoeal disease and an important contributor to early childhood mortality. Cryptosporidium has a single-host life cycle in which both asexual and sexual processes occur in the intestine of infected hosts. Cryptosporidium oocysts are unique in that they mature within the host tissue and are autoinfective They could reset the developmental cycle and maintain infection. Which of these two models applies is a fundamental, yet unanswered, question that has important implications for the disease and the development of drugs and vaccines. We develop molecular markers to observe and analyse the progression of the Cryptosporidium life cycle and use these markers to demonstrate that a block in fertilization limits parasite growth in culture, supporting a model of obligate sexual developmental progression to maintain infection
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