Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum
ABSTRACT The apicomplexan parasite Cryptosporidium is a leading global cause of diarrheal disease, and the infection poses a particularly grave threat to young children and those with weakened immune function. Infection occurs by ingestion of meiotic spores called oocysts, and transmission relies on...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-04-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mbio.03261-22 |
| _version_ | 1797839621741084672 |
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| author | Jayesh Tandel Katelyn A. Walzer Jessica H. Byerly Brittain Pinkston Daniel P. Beiting Boris Striepen |
| author_facet | Jayesh Tandel Katelyn A. Walzer Jessica H. Byerly Brittain Pinkston Daniel P. Beiting Boris Striepen |
| author_sort | Jayesh Tandel |
| collection | DOAJ |
| description | ABSTRACT The apicomplexan parasite Cryptosporidium is a leading global cause of diarrheal disease, and the infection poses a particularly grave threat to young children and those with weakened immune function. Infection occurs by ingestion of meiotic spores called oocysts, and transmission relies on fecal shedding of new oocysts. The entire life cycle thus occurs in a single host and features asexual as well as sexual forms of replication. Here, we identify and locus tag two Apetala 2-type (AP2) transcription factors and demonstrate that they are exclusively expressed in male and female gametes, respectively. To enable functional studies of essential genes in Cryptosporidium parvum, we develop and validate a small-molecule-inducible gene excision system, which we apply to the female factor AP2-F to achieve conditional gene knockout. Analyzing this mutant, we find the factor to be dispensable for asexual growth and early female fate determination in vitro but to be required for oocyst shedding in infected animals in vivo. Transcriptional analyses conducted in the presence or absence of AP2-F revealed that the factor controls the transcription of genes encoding crystalloid body proteins, which are exclusively expressed in female gametes. In C. parvum, the organelle is restricted to sporozoites, and its loss in other apicomplexan parasites leads to blocked transmission. Overall, our development of conditional gene ablation in C. parvum provides a robust method for genetic analysis in this parasite that enabled us to identify AP2-F as an essential regulator of transcription required for oocyst shedding and transmission. IMPORTANCE 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. Transmission occurs via the fecal-oral route by an environmentally resilient spore-like oocyst. Infection takes place in the intestinal epithelium, where parasites initially propagate asexually before transitioning to male and female gametes, with sex leading to the formation of new oocysts. The essential role of sexual development for continuous infection and transmission makes it an attractive target for therapy and prevention. To study essential genes and potential drug targets across the life cycle, we established inducible gene excision for C. parvum. We determined that the female-specific transcription factor AP2-F is not required for asexual growth and early female development in vitro but is necessary for oocyst shedding in vivo. This work enhances the genetic tools available to study Cryptosporidium gene function. |
| first_indexed | 2024-04-09T15:59:48Z |
| format | Article |
| id | doaj.art-9fec4c31937d446991e446cf33744adf |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-04-09T15:59:48Z |
| publishDate | 2023-04-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-9fec4c31937d446991e446cf33744adf2023-04-25T13:04:57ZengAmerican Society for MicrobiologymBio2150-75112023-04-0114210.1128/mbio.03261-22Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvumJayesh Tandel0Katelyn A. Walzer1Jessica H. Byerly2Brittain Pinkston3Daniel P. Beiting4Boris Striepen5Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USADepartment of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USADepartment of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USACenter for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USADepartment of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USADepartment of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USAABSTRACT The apicomplexan parasite Cryptosporidium is a leading global cause of diarrheal disease, and the infection poses a particularly grave threat to young children and those with weakened immune function. Infection occurs by ingestion of meiotic spores called oocysts, and transmission relies on fecal shedding of new oocysts. The entire life cycle thus occurs in a single host and features asexual as well as sexual forms of replication. Here, we identify and locus tag two Apetala 2-type (AP2) transcription factors and demonstrate that they are exclusively expressed in male and female gametes, respectively. To enable functional studies of essential genes in Cryptosporidium parvum, we develop and validate a small-molecule-inducible gene excision system, which we apply to the female factor AP2-F to achieve conditional gene knockout. Analyzing this mutant, we find the factor to be dispensable for asexual growth and early female fate determination in vitro but to be required for oocyst shedding in infected animals in vivo. Transcriptional analyses conducted in the presence or absence of AP2-F revealed that the factor controls the transcription of genes encoding crystalloid body proteins, which are exclusively expressed in female gametes. In C. parvum, the organelle is restricted to sporozoites, and its loss in other apicomplexan parasites leads to blocked transmission. Overall, our development of conditional gene ablation in C. parvum provides a robust method for genetic analysis in this parasite that enabled us to identify AP2-F as an essential regulator of transcription required for oocyst shedding and transmission. IMPORTANCE 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. Transmission occurs via the fecal-oral route by an environmentally resilient spore-like oocyst. Infection takes place in the intestinal epithelium, where parasites initially propagate asexually before transitioning to male and female gametes, with sex leading to the formation of new oocysts. The essential role of sexual development for continuous infection and transmission makes it an attractive target for therapy and prevention. To study essential genes and potential drug targets across the life cycle, we established inducible gene excision for C. parvum. We determined that the female-specific transcription factor AP2-F is not required for asexual growth and early female development in vitro but is necessary for oocyst shedding in vivo. This work enhances the genetic tools available to study Cryptosporidium gene function.https://journals.asm.org/doi/10.1128/mbio.03261-22AP2Cryptosporidiumapicomplexan parasitesintestinesextranscription |
| spellingShingle | Jayesh Tandel Katelyn A. Walzer Jessica H. Byerly Brittain Pinkston Daniel P. Beiting Boris Striepen Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum mBio AP2 Cryptosporidium apicomplexan parasites intestine sex transcription |
| title | Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum |
| title_full | Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum |
| title_fullStr | Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum |
| title_full_unstemmed | Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum |
| title_short | Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum |
| title_sort | genetic ablation of a female specific apetala 2 transcription factor blocks oocyst shedding in cryptosporidium parvum |
| topic | AP2 Cryptosporidium apicomplexan parasites intestine sex transcription |
| url | https://journals.asm.org/doi/10.1128/mbio.03261-22 |
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