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A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum

Cryptosporidium spp., protozoan parasites, are a leading cause of global diarrhea-associated morbidity and mortality in young children and immunocompromised individuals. The limited efficacy of the only available drug and lack of vaccines make it challenging to treat and prevent cryptosporidiosis. T...

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Autores principales: Choudhary, Hadi H., Nava, Maria G., Gartlan, Brina E., Rose, Savannah, Vinayak, Sumiti
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448269/
https://www.ncbi.nlm.nih.gov/pubmed/32843543
http://dx.doi.org/10.1128/mBio.01231-20
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author Choudhary, Hadi H.
Nava, Maria G.
Gartlan, Brina E.
Rose, Savannah
Vinayak, Sumiti
author_facet Choudhary, Hadi H.
Nava, Maria G.
Gartlan, Brina E.
Rose, Savannah
Vinayak, Sumiti
author_sort Choudhary, Hadi H.
collection PubMed
description Cryptosporidium spp., protozoan parasites, are a leading cause of global diarrhea-associated morbidity and mortality in young children and immunocompromised individuals. The limited efficacy of the only available drug and lack of vaccines make it challenging to treat and prevent cryptosporidiosis. Therefore, the identification of essential genes and understanding their biological functions are critical for the development of new therapies. Currently, there is no genetic tool available to investigate the function of essential genes in Cryptosporidium spp. Here, we describe the development of the first conditional system in Cryptosporidium parvum. Our system utilizes the Escherichia coli dihydrofolate reductase degradation domain (DDD) and the stabilizing compound trimethoprim (TMP) for conditional regulation of protein levels in the parasite. We tested our system on the calcium-dependent protein kinase-1 (CDPK1), a leading drug target in C. parvum. By direct knockout strategy, we establish that cdpk1 is refractory to gene deletion, indicating its essentiality for parasite survival. Using CRISPR/Cas9, we generated transgenic parasites expressing CDPK1 with an epitope tag, and localization studies indicate its expression during asexual parasite proliferation. We then genetically engineered C. parvum to express CDPK1 tagged with DDD. We demonstrate that TMP can regulate CDPK1 levels in this stable transgenic parasite line, thus revealing the critical role of this kinase in parasite proliferation. Further, these transgenic parasites show TMP-mediated regulation of CDPK1 levels in vitro and an increased sensitivity to kinase inhibitor upon conditional knockdown. Overall, this study reports the development of a powerful conditional system that can be used to study essential genes in Cryptosporidium.
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spelling pubmed-74482692020-09-02 A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum Choudhary, Hadi H. Nava, Maria G. Gartlan, Brina E. Rose, Savannah Vinayak, Sumiti mBio Research Article Cryptosporidium spp., protozoan parasites, are a leading cause of global diarrhea-associated morbidity and mortality in young children and immunocompromised individuals. The limited efficacy of the only available drug and lack of vaccines make it challenging to treat and prevent cryptosporidiosis. Therefore, the identification of essential genes and understanding their biological functions are critical for the development of new therapies. Currently, there is no genetic tool available to investigate the function of essential genes in Cryptosporidium spp. Here, we describe the development of the first conditional system in Cryptosporidium parvum. Our system utilizes the Escherichia coli dihydrofolate reductase degradation domain (DDD) and the stabilizing compound trimethoprim (TMP) for conditional regulation of protein levels in the parasite. We tested our system on the calcium-dependent protein kinase-1 (CDPK1), a leading drug target in C. parvum. By direct knockout strategy, we establish that cdpk1 is refractory to gene deletion, indicating its essentiality for parasite survival. Using CRISPR/Cas9, we generated transgenic parasites expressing CDPK1 with an epitope tag, and localization studies indicate its expression during asexual parasite proliferation. We then genetically engineered C. parvum to express CDPK1 tagged with DDD. We demonstrate that TMP can regulate CDPK1 levels in this stable transgenic parasite line, thus revealing the critical role of this kinase in parasite proliferation. Further, these transgenic parasites show TMP-mediated regulation of CDPK1 levels in vitro and an increased sensitivity to kinase inhibitor upon conditional knockdown. Overall, this study reports the development of a powerful conditional system that can be used to study essential genes in Cryptosporidium. American Society for Microbiology 2020-08-25 /pmc/articles/PMC7448269/ /pubmed/32843543 http://dx.doi.org/10.1128/mBio.01231-20 Text en Copyright © 2020 Choudhary et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Choudhary, Hadi H.
Nava, Maria G.
Gartlan, Brina E.
Rose, Savannah
Vinayak, Sumiti
A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum
title A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum
title_full A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum
title_fullStr A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum
title_full_unstemmed A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum
title_short A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum
title_sort conditional protein degradation system to study essential gene function in cryptosporidium parvum
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448269/
https://www.ncbi.nlm.nih.gov/pubmed/32843543
http://dx.doi.org/10.1128/mBio.01231-20
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