Transcriptional control of the Cryptosporidium lifecycle

The parasite Cryptosporidium is a leading agent of diarrheal disease in young children, and a cause and consequence of chronic malnutrition. There are no vaccines and only limited treatment options. The parasite infects enterocytes, where it engages in asexual and sexual replication, both of which are essential to continued infection and transmission. However, their molecular mechanisms remain largely unknown. Here we use single-cell RNA sequencing to reveal the gene expression program of the entire C. parvum life cycle in culture and infected animals. Diverging from the prevailing model, we find support for only three intracellular stages: asexual type-I meronts, male gamonts, and female gametes. We uncover a highly organized program for the assembly of components at each stage. Dissecting the underlying regulatory network, we identify the transcription factor Myb-M as the earliest determinant of male fate, in an organism that lacks genetic sex determination. Conditional expression of this factor overrides the developmental program and induces widespread maleness, while conditional deletion ablates male development. Both have a profound impact on the infection. A large set of stage-specific genes now provides the opportunity to understand, engineer, and disrupt parasite sex and life cycle progression to advance the development of vaccines and treatments. Bulk and single-cell RNA sequencing (scRNA-seq) data were generated in this study. Samples for scRNA-seq and stage-specific bulk RNA sequencing were flow sorted to enrich for infected cells. For bulk analysis of stage-specific reporters, asexual and female datasets were already available (GEO:GSE129267) while the male in vitro dataset, consisting of four biological replicates, was produced in this study. For bulk analysis of Myb-M gain-of-function mutants, three biological replicates were used for each strain and condition collected at 30 hours after 6 hours of vehicle or Shield-1 treatment. For scRNA-seq analysis, infected cells were captured from culture or a mouse. In vitro samples were collected at 24, 36, 42, 46, and 54 hours while the in vivo sample was collected from an acutely infected mouse on day 6.