Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum

Tandel, Jayesh; Walzer, Katelyn A.; Byerly, Jessica H.; Pinkston, Brittain; Beiting, Daniel P.; Striepen, Boris

doi:10.1128/mbio.03261-22

Cited by 15 publications

(5 citation statements)

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“…4 B ) consistent with Cre-mediated excision. We also noted some leaky Cre activity in the absence of rapamycin as previously described ( 34 ). Infected cultures grown in the presence or absence of rapamycin for 48 h, were fixed and imaged.…”

Section: Resultssupporting

confidence: 70%

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Genetic crosses within and between species of Cryptosporidium

Shaw,

Cohn,

Baptista

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Resultssupporting

confidence: 70%

“…We recently demonstrated the use of dimerizable Cre recombinase (DiCre) for rapamycin induced gene ablation; however, introducing loxP sites and recombinase in a single transfection is challenging (see extended technical discussion in ref. 34 ). To overcome this, we devised a model in which we cross a Cre driver and a floxed strain ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Genetic crosses within and between species of Cryptosporidium

Shaw,

Cohn,

Baptista

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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“…The seminal work of English et al (2022) provided insights into the infection kinetics of C. parvum, where it was demonstrated that the parasite must undergo three cycles of asexual replication before sexual development will occur (42). Within the transwell plate, fertilisation is very limited which results in cessation of C. parvum infection after three days post-infection (20,43). The findings of the present work suggest that one cycle of successful sexual replication occurred within the microfluidic device, and excystation of oocysts produced in vitro occurred within the microchannel.…”

Section: Discussionmentioning

confidence: 99%

A pumpless and tubeless microfluidic device enables extendedin vitrodevelopment ofCryptosporidium parvum

Gunasekera,

Thierry,

Cheah

et al. 2024

Preprint

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The enteric parasiteCryptosporidiumremains a treatment challenge for drinking water utilities globally due to its resistance to chlorine disinfection. However, the lack of anin vitroculture system forCryptosporidiumthat is both cost-effective and reliable remains a key bottleneck inCryptosporidiumresearch. Here we report that the microfluidic culture of HCT-8 cells under fluid shear stress enables the extended development ofCryptosporidium parvum. Specifically, the growth ofC. parvumin a user-friendly pumpless microfluidic device was assessed using immunofluorescence assays, scanning electron microscopy and quantitative PCR, which revealed that development peaked at six days post-infection but continued for ten days in total. Oocysts produced within the microfluidic device were infective to fresh HCT-8 monolayers, however these oocysts were only present at low levels. We anticipate that such microfluidic approaches will facilitate a wide range ofin vitrostudies onCryptosporidiumand may have the potential to be further developed as a routine infectivity assessment tool for the water industry.

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“…Despite extensive research on ApiAP2 TFs in apicomplexan parasites such as Plasmodium spp., T. gondii, and Cryptosporidium [35][36][37][38][39][40], their function in Eimeria pp. has not been studied to date.…”

Section: Discussionmentioning

confidence: 99%

Comparative transcriptome profiling of Eimeria tenella in various developmental stages and functional analysis of an ApiAP2 transcription factor exclusively expressed during sporogony

et al. 2023

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Background The apicomplexan parasites Eimeria spp. are the causative agents of coccidiosis, a disease with a significant global impact on the poultry industry. The complex life cycle of Eimeria spp. involves exogenous (sporogony) and endogenous (schizogony and gametogony) stages. Unfortunately, the genetic regulation of these highly dynamic processes, particularly for genes involved in specific developmental phases, is not well understood. Methods In this study, we used RNA sequencing (RNA-Seq) analysis to identify expressed genes and differentially expressed genes (DEGs) at seven time points representing different developmental stages of Eimeria tenella. We then performed K-means clustering along with co-expression analysis to identify functionally enriched gene clusters. Additionally, we predicted apicomplexan AP2 transcription factors in E. tenella using bioinformatics methods. Finally, we generated overexpression and knockout strains of ETH2_0411800 to observe its impact on E. tenella development. Results In total, we identified 7329 genes that are expressed during various developmental stages, with 3342 genes exhibiting differential expression during development. Using K-means clustering along with co-expression analysis, we identified clusters functionally enriched for oocyte meiosis, cell cycle, and signaling pathway. Among the 53 predicted ApiAP2 transcription factors, ETH2_0411800 was found to be exclusively expressed during sporogony. The ETH2_0411800 overexpression and knockout strains did not exhibit significant differences in oocyst size or output compared to the parental strain, while the resulting ETH2_0411800 knockout parasite showed a relatively small oocyst output. Conclusions The findings of our research suggest that ETH2_0411800 is not essential for the growth and development of E. tenella. Our study provides insights into the gene expression dynamics and is a valuable resource for exploring the roles of transcription factor genes in regulating the development of Eimeria parasites. Graphical Abstract

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Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum

Abstract: The parasite Cryptosporidium infects millions of people worldwide each year, leading to life-threatening diarrheal disease in young children and immunosuppressed individuals. There is no vaccine and only limited treatment.

Cited by 15 publications

References 65 publications

Genetic crosses within and between species of Cryptosporidium

Genetic crosses within and between species of Cryptosporidium

A pumpless and tubeless microfluidic device enables extendedin vitrodevelopment ofCryptosporidium parvum

Comparative transcriptome profiling of Eimeria tenella in various developmental stages and functional analysis of an ApiAP2 transcription factor exclusively expressed during sporogony

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