Sebastian Shaw

Infection with Cryptosporidium is a leading cause of diarrheal disease and early childhood mortality. This apicomplexan parasite undergoes asexual and sexual replication within the same host, and recent studies have shown an intrinsic developmental program of obligate transition to male and female gametes and sex. While factors were identified that control male fate and development, how female gene expression is orchestrated remains largely unknown. Here, we use the Cryptosporidium Single Cell Atlas to discover an RNA-binding protein (F-RBP) as one of the earliest markers of female identity in C. parvum . Reporter parasites engineered based on this gene allowed us to calibrate transcriptional pseudotime against the real time of female development, revealing a significant window of transcriptional fate ambiguity. While F-RBP is an early transcript, the protein persists throughout female development and into the zygote. Conditional ablation of the F-RBP gene showed it to be dispensable for sex determination and early female development in vitro . However, the gene is essential in vivo, and its loss results in rapid cure. Cell biological experiments link this loss to the production of oocysts that release sporozoites incapable of host-cell invasion. F-RBP binds transcripts highly expressed in the female gamete, enriched for a sequence motif recognized by a Y-box protein family member, and forms mRNA-protein complexes in late females akin to processing or P bodies. We propose F-RBP’s essential role to be in the regulation of long-term homeostasis of maternally inherited RNA required for sporozoite infectivity. IMPORTANCE: The parasite Cryptosporidium is responsible for millions of cases of severe diarrheal disease in young children, and the infection is both a consequence of and a risk factor for chronic malnutrition. There are no vaccines available, and a single approved treatment is largely ineffective. Infection relies on ingestion of the oocyst, an environmentally stable spore, which contains an invasive form of the parasite, the sporozoite. Oocysts and sporozoites are the products of parasite sex and the fusion of female and male Cryptosporidium parasites. Here, we report the critical role of an RNA-binding protein in female mRNA homeostasis. Maternal transcripts orchestrate the complex process of sexual replication and oocyst formation. Loss of the protein results in spores that fail to initiate the next round of the life cycle, and conditional ablation using a small-molecule trigger quickly cures infected animals, demonstrating the essential role of parasite sex in continued infection.

Infection with Cryptosporidium is a leading cause of diarrheal disease and early childhood mortality. This apicomplexan parasite undergoes asexual and sexual replication within the same host and recent studies have shown an intrinsic developmental program of obligate transition to male and female gametes and sex. While factors were identified that control male fate and development, how female gene expression is orchestrated remains largely unknown. Here we use the Cryptosporidium Single Cell Atlas to discover an RNA binding protein (F-RBP) as one of the earliest markers of female identity. Reporter parasites engineered based on this gene allowed us to calibrate transcriptional pseudotime against the real time of female development revealing a significant window of transcriptional fate ambiguity. While F-RBP is an early transcript, the protein persists throughout female development and into the zygote. Conditional ablation of the F-RBP gene showed it to be dispensable for sex determination and early female development in vitro . However, the gene is essential in vivo and its loss results in rapid cure. Cell biological experiments link this loss to the production of sterile oocysts which release sporozoites incapable of host cell invasion. F-RBP binds transcripts highly expressed in the female gamete enriched for a YBOX primary sequence motif and forms mRNA protein complexes in late females akin to processing or P bodies. We propose F-RBP’s essential role to be in the regulation of long-term homeostasis of maternally inherited RNA required for sporozoite infectivity. Importance: The parasite Cryptosporidium is responsible for millions of cases of severe diarrheal disease in young children and is both a consequence of, and a risk factor for, chronic malnutrition. There are no vaccines available and a single approved treatment is largely ineffective. Infection relies on ingestion of the oocyst, an environmentally stable spore, which contains an invasive form of the parasite, the sporozoite. Oocyst and sporozoites are the product of parasite sex and the fusion of female and male Cryptosporidium parasites. Here we report the critical role of an RNA- binding protein in female mRNA homeostasis. Maternal transcripts orchestrate the complex process of sexual replication and oocyst formation. Loss of the protein results in sterile spores that fail to initiate the next round of the lifecycle, and conditional ablation using a small molecule trigger quickly cures infected animals demonstrating the essential role of parasite sex in continued infection.

The parasite Cryptosporidium is a leading cause of life-threatening diarrhoeal disease, and effective treatment is not available. Clofazimine, an antimicrobial used for treatment of leprosy and tuberculosis, was found to have potent anti- Cryptosporidium activity but it failed in a human trial. This was attributed to poor bioavailability. Here we observed differential clofazimine susceptibility among C. parvum parasite isolates, which we exploit to identify a single genomic locus encoding the type II NADH dehydrogenase (NDH2) in an unbiased genetic cross. Targeted genetic ablation of ndh2 resulted in high-level clofazimine resistance and biochemical studies demonstrated NDH2-mediated electron transfer to clofazimine. Through genomic analyses, we uncovered heterogeneity at the ndh2 locus for C. parvum and C. hominis , and widespread carriage of a conserved attenuated allele across multiple continents. This heterogeneity allows parasites genomically linked through frequent sexual recombination to adjust to changing NDH2 requirements and predisposes Cryptosporidium to evade clofazimine treatment.

The parasite Cryptosporidium is a leading cause of life-threatening diarrheal disease, and effective treatment is not available. The discovery of potent anti- Cryptosporidium activity of clofazimine, offered the opportunity to repurpose a drug already used to treat leprosy and tuberculosis. However, clofazimine failed in a human trial, which was attributed to poor bioavailability. Here, we observed differential susceptibility among parasite isolates which we exploit to map the mode of action to type II NADH dehydrogenase (NDH2) in an unbiased genetic cross. Targeted genetic ablation of NDH2 resulted in profound clofazimine resistance, and biochemical studies demonstrated NDH2 mediated electron transfer to clofazimine. Through genomic analyses we uncovered heterogeneity at the NDH2 locus for C. parvum and C. hominis and widespread carriage of a conserved attenuated allele across multiple continents. This heterogeneity allows parasites genomically linked through frequent sexual recombination to adjust to changing NDH2 requirements and predisposes Cryptosporidium to evade clofazimine treatment.

The relationship between parasite genotype and pathogenesis is largely unknown for Cryptosporidium , a leading cause of diarrheal disease in children. An array of parasites with similar genomes produces varied disease outcomes in different hosts. Here, we isolate and characterize Cryptosporidium parvum strains that show marked differences in virulence and persistence in mice. Taking advantage of the sexual lifecycle of this eukaryotic pathogen, we use genetic crosses to discover the underlying chromosomal loci. Whole-genome sequencing and bulk segregant analysis of infection selected progeny mapped three loci on chromosomes 2, 6, and 7 associated with the ability to colonize and persist in mice and the positions of drug resistance genes. The chromosome 6 locus encodes the hyper-polymorphic surface glycoprotein GP60. Reverse genetic studies in both parental strains demonstrate that GP60 controls parasite burden and virulence, but not persistence, and reveal the dominance of the less virulent allele, suggesting it restricts virulence.

Cryptosporidium is an enteric pathogen and a prominent cause of diarrheal disease worldwide. Control of Cryptosporidium requires CD4+ T cells, but how protective CD4+ T cell responses are generated is poorly understood. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to understand the basis for CD4+ T cell priming and effector function. These studies revealed that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node but differentiate into Th1 cells in the gut to provide local parasite control. Although type 1 conventional dendritic cells (cDC1s) were dispensable for CD4+ T cell priming, they were required for CD4+ T cell gut homing and were a source of IL-12 at the site of infection that promoted local production of IFN-γ. Thus, cDC1s have distinct roles in shaping CD4+ T cell responses to an enteric infection: first, to promote gut homing from the mesLN, and second, to drive effector responses in the intestine.

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.

Cryptosporidium is an enteric pathogen that is a prominent cause of diarrheal disease. Control of this infection requires CD4 ⁺ T cells, though the processes that lead to T cell-mediated resistance have been difficult to assess. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to dissect the early events that influence CD4 ⁺ T cell priming and effector function. These studies highlight that parasite-specific CD4 ⁺ T cells are primed in the draining mesenteric lymph node (mesLN) and differentiate into Th1 cells in the gut, where they mediate IFN-γ-dependent control of the infection. Although type 1 conventional dendritic cells (cDC1s) were not required for initial priming of CD4 ⁺ T cells, cDC1s were required for CD4 ⁺ T cell expansion and gut homing. cDC1s were also a major source of IL-12 that was not required for priming but promoted full differentiation of CD4 ⁺ T cells and local production of IFN-γ. Together, these studies reveal distinct roles for cDC1s in shaping CD4 ⁺ T cell responses to enteric infection: first to drive early expansion in the mesLN and second to drive effector responses in the gut. Summary: Cryptosporidium parasites that express model antigens were generated to dissect how parasite-specific CD4 ⁺ T cells are primed and mediate effector functions required to control this enteric pathogen. cDC1s produced IL-12p40 and were required for early expansion and gut homing of CD4 ⁺ T cells. However, IL-12p40 was only required for the development of Th1 CD4 ⁺ T cell effector function in the gut.

Parasites and their hosts are engaged in rapid 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 C. parvum, a parasite of cattle and humans, and C. 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. Significance statement: The parasite Cryptosporidium is a leading cause of diarrheal disease. While infection is common all around the world, young children experiencing malnutrition are impacted most profoundly, and the disease is an important contributor to early childhood mortality. This study experimentally demonstrates that different strains and even species of Cryptosporidium can recombine their genomes through sex. The progeny of such genetic crosses shows combined features of both parents, with resistance to multiple drugs being one example. Sex thus provides a critical mechanism for the parasite to rapidly adapt to changing environments and hosts. Genetic crosses as an experimental tool may also be harnessed in the future to discover the genes underlying differences in virulence, drug sensitivity, and immunogenicity between parasite isolates.