Dataset Information

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

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.

SUBMITTER: Witola WH

PROVIDER: S-EPMC5665856 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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

Witola William H WH Zhang Xuejin X Kim Chi Yong CY

International journal for parasitology 20170609 13

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 endea ...[more]

PMID: 28606696

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Project description:BackgroundHundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.ResultsThe C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC₅₀ values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.ConclusionsIdentification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.

Dataset Information

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

Publications

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

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