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

William H. Witola, Xuejin Zhang, Chi Yong Kim

Research output: Research - peer-reviewArticle

Abstract

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.

LanguageEnglish (US)
Pages867-874
Number of pages8
JournalInternational Journal for Parasitology
Volume47
Issue number13
DOIs
StatePublished - Nov 1 2017

Fingerprint

Gene Knockdown Techniques
Cryptosporidium parvum
L-Lactate Dehydrogenase
Morpholinos
Genes
Growth and Development
Parasites
Pharmaceutical Preparations
In Vitro Techniques
human PRMT2 protein
nitazoxanide
Child Nutrition Disorders
Cryptosporidium
Zoonoses
Protein C
Cytosol
Down-Regulation
Growth
Infection
Therapeutics

Keywords

  • Cryptosporidium parvum
  • Gene knockdown
  • Lactate dehydrogenase function
  • Phosphorodiamidate morpholino oligomers

ASJC Scopus subject areas

  • Parasitology
  • Infectious Diseases

Cite this

Targeted gene knockdown validates the essential role of lactate dehydrogenase in Cryptosporidium parvum. / Witola, William H.; Zhang, Xuejin; Kim, Chi Yong.

In: International Journal for Parasitology, Vol. 47, No. 13, 01.11.2017, p. 867-874.

Research output: Research - peer-reviewArticle

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