Effect of Metabolic Enzymes on Amylopectin Content and Infectivity of Cryptosporidium parvum
Hartman, Angela Danielle
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Amylopectin granules in Apicomplexan protozoa are hypothesized to be used as an energy source to aid the parasites in surviving in the environment allow latent stages to excyst and release infective stages, allow them to be mobile, invade host cells, and to continue their life cycle. The objective of this project was to determine if parasite glycolytic enzymes: alpha-amylase, amyloglucosidase, enolase, lactate dehydrogenase, and phosphorylase could be used to decrease amylopectin stores and subsequently infectivity of Cryptosporidium parvum oocysts/sporozoites in both fresh oocysts and stored oocysts. In addition, glycolytic enzymes and substrates: glucose, glucose-1-phosphate, and glycogen synthase were investigated to determine if they can be used to increase amylopectin stores and thus increase infectivity to aid in detection/storage of oocysts. Oocysts of Cryptosporidium parvum were suspended in 1mg/ml glycolytic enzymes or substrates (except glucose - 0.05M and glycogen synthase - 1U/ml) and electroporated. Oocysts were incubated at 37°C for one hour to allow treatments to react with amylopectin followed by incubation on HCT-8 cells for 24 hours for infection. Real-time PCR and immunohistochemistry were performed to determine the effect of the enzymes on infectivity. An amylopectin assay and excystation assay was performed to determine if the enzymes degraded amylopectin and if decreased amylopectin reduced excystation. Alpha amylase and amyloglucosidase had the greatest impact on reducing both amylopectin and infectivity of fresh oocysts with reductions of 99.5% and 99.1% in infective oocysts, respectively (P<0.05). These results suggest that amylopectin may be an important factor in infection, although further research is needed. In stored oocysts, enzymes significantly reduced amylopectin content but not infectivity. In fresh oocysts, amylopectin content was correlated to excystation and infectivity with a decrease in amylopectin correlating to decreased excystation and infectivity. In contrast, there was no direct correlation for stored oocysts. When glucose, glucose-1-phosphate, or glycogen synthase was used to increase infectivity, results show that glycogen synthase had little effect, but glucose and glucose-1-phosphate significantly increased amylopectin content, excystation, and infectivity. In conclusion, amylopectin may be an important polysaccharide store of Cryptosporidium parasites to cause infection by allowing excystation of the oocysts to release infective sporozoites.
- Doctoral Dissertations