Genetic crosses within and between species of Cryptosporidium

Abstract

Parasites and their hosts are engaged in reciprocal coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium , genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach, multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing Cryptosporidium parvum, a parasite of cattle and humans, and Cryptosporidium tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity.

Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-γ on parasite restriction

“…Recent studies on the population structure of Cryptosporidium revealed adaptation to humans as a host and it seems likely that this was dependent on the development of species-specific immune evasion strategies [ 60 – 62 ]. As such, the ability to compare Cryptosporidium species and strains that differ in virulence may provide new insights into host-specific interference with IFN-γ signalling while the ability to conduct genetic crosses between strains [ 63 ] offers the opportunity to uncover parasite genes and proteins that underlie these differences.…”

Cryptosporidium Genomics — Current Understanding, Advances, and Applications

“…The protocol involves oocyst sorting, lysis, genome amplification with multiple displacement amplification (MDA), and sequencing with short-read Illumina [ 16 ]. This technique has recently been modified to utilize long-read ONT sequencing [ 17 ••, 18 ]. Given the obligately sexual nature of Cryptosporidium and the existence of four related haploid sporozoites within an oocyst, single-oocyst sequencing is also a promising technique for studies of diversity within a single infection [ 16 ].…”

“…Given the obligately sexual nature of Cryptosporidium and the existence of four related haploid sporozoites within an oocyst, single-oocyst sequencing is also a promising technique for studies of diversity within a single infection [ 16 ]. Single-oocyst sequencing also creates an avenue for studying diversity and recombination events within a single oocyst [ 17 ••].…”

“…Recently, a genetic cross was reported not only within C. parvum but also between C. parvum and C. tyzzeri [ 17 ••]. In addition to being a major genetics breakthrough for Cryptosporidium research, this finding, combined with the recombination and introgression observations above, raises questions regarding the definition of what constitutes a species in Cryptosporidium .…”

“…Several selectable genetic markers are now available [ 17 ••, 46 , 51 ••], and this development has permitted genetic crosses [ 17 ••, 51 ••] in immunocompromised mice. This advance is of considerable significance.…”