PROJECT SUMMARY/ABSTRACT
Cryptosporidium is a wide-spread enteric pathogen that causes severe diarrheal disease in
immunocompromised patients and infants, especially in the developing world. There are more than 20
described species of Cryptosporidium some of which show narrow host ranges while others are quite broad. C.
parvum has a relatively broad host range infecting many agricultural animals and causing zoonotic infections in
humans. C. parvum is further divided into subtypes that undergo recombination in the wild often resulting in in
differences in host range, infectivity, and virulence. The factors that control host range of C. parvum as well as
their pathogenicity in different hosts are largely unknown, presenting a major gap in our understanding of this
important group of parasites. In preliminary studies, we have defined major virulence differences between
common laboratory variants of C. parvum during infection in immunocompromised mice. High-virulence
isolates are associated with elevated oocyst shedding and lethality, while low-virulence isolates cause only
mild, non-lethal infections. Whole genome sequencing identified numerous single nucleotide polymorphisms in
candidate genes that likely control these phenotypic differences. We recently developed a stem-cell derived
system for culturing differentiated intestinal enterocytes that allows complete development of C. parvum in
vitro, including production of viable oocysts. In preliminary studies, we tagged C. parvum isolates with
fluorescent reporters using CRISPR/Cas9 gene editing and used these lines to conduct genetic crosses in
vitro. Analysis of the recombinant progeny from crossed lines demonstrates that meiosis results in Mendelian
chromosome reassortment. In the proposed studies, we will cross fluorescently tagged virulent isolates with
avirulent isolates in vitro, sort recombinant progeny using FACS, and expand them in mice to select for genes
associated with increased fitness. The contributing loci will be mapped using Bulk Segregant Analysis to define
candidate genes that will be confirmed by CRISPR/Cas editing. These studies should lead to a molecular
understanding of pathogenicity of C. pavrum and may identify genes responsible for mediating infectivity and
host-range.