In Vitro Enrichment of Phosphorylated Proteins From Synchronously Excysted Cryptosporidium Parvum
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Cryptosporidium is an enteric protozoan parasite that causes cryptosporidiosis and there is no effective drug treatment available. Two studies were conducted. In the first, a novel protocol was developed for the rapid in vitro excystation of Cryptosporidium parvum. Rapid excystation (release of infectious sporozoites from the environmental oocyst stage) is necessary to study molecular dynamics during the early stages of Cryptosporidium development. The developed excystation assay (Joshi assay) used an HCl (pH 2.5) oocyst pretreatment instead of the bleach pretreatment that is more frequently used in excystation assays. The Joshi assay achieved >90% excystation in 10 min. This rapid, reproducible, and synchronous in vitro excystation assay mimics the in vivo conditions in a mammal. In the second study, phosphorylated and non-phosphorylated proteins were selectively enriched from non-excysted and excysted C. parvum oocysts and separated using 2 dimensional gel electrophoresis. This is the first study that enriched phosphorylated proteins from C.parvum.