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 36h 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 56h 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.
Highlights
Cryptosporidium parvum is a highly prevalent zoonotic and anthroponotic apicomplexan protozoan of medical and veterinary significance
In the present study, we endeavoured to adapt the use of a phosphorodiamidate morpholino oligomers antisense approach to develop a targeted gene knockdown assay to study and validate gene function in C. parvum
We found that the standard off-target control, C. parvum lactate dehydrogenase gene (CpLDH) and C. parvum putative arginine n-methyltransferase (CpAMT) vivo morpholino had IC50 values in HCT-8 cells of 32 ± 5.3 lM, ± 3.8 lM and ± 4.6 lM, respectively
Summary
Cryptosporidium parvum is a highly prevalent zoonotic and anthroponotic apicomplexan protozoan of medical and veterinary significance. It causes a serious diarrheal syndrome (cryptosporidiosis) in calves, lambs and goat kids, resulting in poor growth rates and high neonatal mortality (de Graaf et al, 1999; Jex and Gasser, 2009; Karanis et al, 2010). (C. parvum together with Cryptosporidium hominis) are the number two major cause of diarrheal disease in young children resulting in significant childhood morbidity and mortality in developing countries (Checkley et al, 2015). A compounding problem of Cryptosporidium infection is the unavailability of fully effective drugs or vaccines against it
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