A Conditional Protein Degradation System To Study Essential Gene Function in <named-content content-type="genus-species">Cryptosporidium parvum</named-content>

ABSTRACT 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 cryptospori...

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Autores principales:	Hadi H. Choudhary, Maria G. Nava, Brina E. Gartlan, Savannah Rose, Sumiti Vinayak
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	CRISPR/Cas9 conditional system Cryptosporidium apicomplexan parasite drug targets molecular genetics Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a4543fb2658741efa358b34c8a7166ac
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spelling	oai:doaj.org-article:a4543fb2658741efa358b34c8a7166ac2021-11-15T15:56:44ZA Conditional Protein Degradation System To Study Essential Gene Function in <named-content content-type="genus-species">Cryptosporidium parvum</named-content>10.1128/mBio.01231-202150-7511https://doaj.org/article/a4543fb2658741efa358b34c8a7166ac2020-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01231-20https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE Cryptosporidium parvum and Cryptosporidium hominis are leading pathogens responsible for diarrheal disease (cryptosporidiosis) and deaths in infants and children below 5 years of age. There are no effective treatment options and no vaccine for cryptosporidiosis. Therefore, there is an urgent need to identify essential gene targets and uncover their biological function to accelerate the development of new and effective anticryptosporidial drugs. Current genetic tool allows targeted disruption of gene function but leads to parasite lethality if the gene is essential for survival. In this study, we have developed a genetic tool for conditional degradation of proteins in Cryptosporidium spp., thus allowing us to study the function of essential genes. Our conditional system expands the molecular toolbox for Cryptosporidium, and it will help us to understand the biology of this important human diarrheal pathogen for the development of new drugs and vaccines.Hadi H. ChoudharyMaria G. NavaBrina E. GartlanSavannah RoseSumiti VinayakAmerican Society for MicrobiologyarticleCRISPR/Cas9conditional systemCryptosporidiumapicomplexan parasitedrug targetsmolecular geneticsMicrobiologyQR1-502ENmBio, Vol 11, Iss 4 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR/Cas9 conditional system Cryptosporidium apicomplexan parasite drug targets molecular genetics Microbiology QR1-502
spellingShingle	CRISPR/Cas9 conditional system Cryptosporidium apicomplexan parasite drug targets molecular genetics Microbiology QR1-502 Hadi H. Choudhary Maria G. Nava Brina E. Gartlan Savannah Rose Sumiti Vinayak A Conditional Protein Degradation System To Study Essential Gene Function in <named-content content-type="genus-species">Cryptosporidium parvum</named-content>
description	ABSTRACT 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. IMPORTANCE Cryptosporidium parvum and Cryptosporidium hominis are leading pathogens responsible for diarrheal disease (cryptosporidiosis) and deaths in infants and children below 5 years of age. There are no effective treatment options and no vaccine for cryptosporidiosis. Therefore, there is an urgent need to identify essential gene targets and uncover their biological function to accelerate the development of new and effective anticryptosporidial drugs. Current genetic tool allows targeted disruption of gene function but leads to parasite lethality if the gene is essential for survival. In this study, we have developed a genetic tool for conditional degradation of proteins in Cryptosporidium spp., thus allowing us to study the function of essential genes. Our conditional system expands the molecular toolbox for Cryptosporidium, and it will help us to understand the biology of this important human diarrheal pathogen for the development of new drugs and vaccines.
format	article
author	Hadi H. Choudhary Maria G. Nava Brina E. Gartlan Savannah Rose Sumiti Vinayak
author_facet	Hadi H. Choudhary Maria G. Nava Brina E. Gartlan Savannah Rose Sumiti Vinayak
author_sort	Hadi H. Choudhary
title	A Conditional Protein Degradation System To Study Essential Gene Function in <named-content content-type="genus-species">Cryptosporidium parvum</named-content>
title_short	A Conditional Protein Degradation System To Study Essential Gene Function in <named-content content-type="genus-species">Cryptosporidium parvum</named-content>
title_full	A Conditional Protein Degradation System To Study Essential Gene Function in <named-content content-type="genus-species">Cryptosporidium parvum</named-content>
title_fullStr	A Conditional Protein Degradation System To Study Essential Gene Function in <named-content content-type="genus-species">Cryptosporidium parvum</named-content>
title_full_unstemmed	A Conditional Protein Degradation System To Study Essential Gene Function in <named-content content-type="genus-species">Cryptosporidium parvum</named-content>
title_sort	conditional protein degradation system to study essential gene function in <named-content content-type="genus-species">cryptosporidium parvum</named-content>
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/a4543fb2658741efa358b34c8a7166ac
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A Conditional Protein Degradation System To Study Essential Gene Function in <named-content content-type="genus-species">Cryptosporidium parvum</named-content>

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