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Targeted gene knockdown validates the essential role of lactate dehydrogenase in Cryptosporidium parvum

Cryptosporidium parvum is a zoonotic protozoan that can cause a life-threatening gastrointestinal syndrome in children and in immunocompromised adults. Currently, the only approved drug for treatment of Cryptosporidium infections in humans is nitazoxanide, but it is not effective in immunocompromise...

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Autores principales: Witola, William H., Zhang, Xuejin, Kim, Chi Yong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665856/
https://www.ncbi.nlm.nih.gov/pubmed/28606696
http://dx.doi.org/10.1016/j.ijpara.2017.05.002
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author Witola, William H.
Zhang, Xuejin
Kim, Chi Yong
author_facet Witola, William H.
Zhang, Xuejin
Kim, Chi Yong
author_sort Witola, William H.
collection PubMed
description Cryptosporidium parvum is a zoonotic protozoan that can cause a life-threatening gastrointestinal syndrome in children and in immunocompromised adults. Currently, the only approved drug for treatment of Cryptosporidium infections in humans is nitazoxanide, but it is not effective in immunocompromised individuals or in children with malnutrition. This is compounded by the lack of genetic methods for studying and validating potential drug targets in the parasite. Therefore, in this study, we endeavoured to adapt the use of a phosphorodiamidate morpholino oligomer (morpholino) antisense approach to develop a targeted gene knockdown assay for use in C. parvum. We show that morpholinos, at non-toxic concentrations, are rapidly internalised by both C. parvum and host cells (HCT-8), and distribute diffusely throughout the cytosol. Using morpholinos to separately target C. parvum lactate dehydrogenase and putative arginine n-methyltransferase genes, within 36 h of in vitro culture, we achieved over 10-fold down-regulation of the respective encoded proteins in C. parvum. Pursuant to this, we observed that knockdown of C. parvum lactate dehydrogenase produced a dramatic reduction in intracellular growth and development of C. parvum by 56 h of culture. On the other hand, C. parvum putative arginine n-methyltransferase knockdown did not appear to have any effect on parasite growth, but nevertheless provided the proof-of-principle that the morpholino knockdown assay in C. parvum was consistent. Together, our findings present a gene regulation approach for interrogating gene function in C. parvum in vitro, and further provide genetic evidence for the essential role of C. parvum lactate dehydrogenase in fueling the growth and development of intracellular C. parvum.
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spelling pubmed-56658562017-11-09 Targeted gene knockdown validates the essential role of lactate dehydrogenase in Cryptosporidium parvum Witola, William H. Zhang, Xuejin Kim, Chi Yong Int J Parasitol Article Cryptosporidium parvum is a zoonotic protozoan that can cause a life-threatening gastrointestinal syndrome in children and in immunocompromised adults. Currently, the only approved drug for treatment of Cryptosporidium infections in humans is nitazoxanide, but it is not effective in immunocompromised individuals or in children with malnutrition. This is compounded by the lack of genetic methods for studying and validating potential drug targets in the parasite. Therefore, in this study, we endeavoured to adapt the use of a phosphorodiamidate morpholino oligomer (morpholino) antisense approach to develop a targeted gene knockdown assay for use in C. parvum. We show that morpholinos, at non-toxic concentrations, are rapidly internalised by both C. parvum and host cells (HCT-8), and distribute diffusely throughout the cytosol. Using morpholinos to separately target C. parvum lactate dehydrogenase and putative arginine n-methyltransferase genes, within 36 h of in vitro culture, we achieved over 10-fold down-regulation of the respective encoded proteins in C. parvum. Pursuant to this, we observed that knockdown of C. parvum lactate dehydrogenase produced a dramatic reduction in intracellular growth and development of C. parvum by 56 h of culture. On the other hand, C. parvum putative arginine n-methyltransferase knockdown did not appear to have any effect on parasite growth, but nevertheless provided the proof-of-principle that the morpholino knockdown assay in C. parvum was consistent. Together, our findings present a gene regulation approach for interrogating gene function in C. parvum in vitro, and further provide genetic evidence for the essential role of C. parvum lactate dehydrogenase in fueling the growth and development of intracellular C. parvum. Elsevier Science 2017-11 /pmc/articles/PMC5665856/ /pubmed/28606696 http://dx.doi.org/10.1016/j.ijpara.2017.05.002 Text en © 2017 The Author(s) http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Witola, William H.
Zhang, Xuejin
Kim, Chi Yong
Targeted gene knockdown validates the essential role of lactate dehydrogenase in Cryptosporidium parvum
title Targeted gene knockdown validates the essential role of lactate dehydrogenase in Cryptosporidium parvum
title_full Targeted gene knockdown validates the essential role of lactate dehydrogenase in Cryptosporidium parvum
title_fullStr Targeted gene knockdown validates the essential role of lactate dehydrogenase in Cryptosporidium parvum
title_full_unstemmed Targeted gene knockdown validates the essential role of lactate dehydrogenase in Cryptosporidium parvum
title_short Targeted gene knockdown validates the essential role of lactate dehydrogenase in Cryptosporidium parvum
title_sort targeted gene knockdown validates the essential role of lactate dehydrogenase in cryptosporidium parvum
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665856/
https://www.ncbi.nlm.nih.gov/pubmed/28606696
http://dx.doi.org/10.1016/j.ijpara.2017.05.002
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