Effect of metabolic enzymes on amylopectin content and infectivity of Cryptosporidium parvum

• Main Author: Hartman, Angela D
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2006
• Subjects: Biochemistry; Microbiology
• ISBN: 9780542970900; 0542970902

Many parasites belonging to the phylum Apicomplexa have the ability to cause illness in both humans and other animal species. The majority of these apicomplexans contain amylopectin granules for use as energy throughout their life cycles. It has been hypothesized that amylopectin granules in Apicomplexan protozoa are used as an energy source to aid the parasites in surviving in the environment in the oocyst stage, to allow latent stages to excyst and release infective stages, to allow them to be mobile to access infective sites, invade host cells, and to convert to other life stages 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 in Cryptosporidium parvum oocysts/sporozoites and thereby reduce infectivity and potentially be used as a control method in both freshly excreted oocysts and those that have been surviving in the environment. 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 and storage of oocysts. Oocysts of Cryptosporidium parvum were suspended in a solution containing 1mg/ml glycolytic enzymes or substrates (with the exception of glucose used at 0.05M and glycogen synthase at 1U/ml) and electroporated to allow the enzymes to enter the oocysts. The oocysts were incubated at 37°C for 1 hour to allow the enzyme or substrate treatments to react with amylopectin granules. The oocysts were incubated on HCT-8 cells for 24 hours to allow infection. Real-time PCR and immunohistochemistry were performed to determine the effect of the enzymes on infectivity of sporozoites released from treated oocysts. In addition, an amylopectin assay and excystation assay was performed to determine if the enzymes were able to degrade amylopectin and if a decrease in amylopectin reduced the amount of energy available for excystation, respectively. Alpha amylase and amyloglucosidase had the greatest impact on reducing both the amount of amylopectin and the infectivity of fresh oocysts with reductions of 99.6% and 99.7% in infective oocysts, respectively (p<0.05). These results suggest that amylopectin may indeed be an important factor that parasites use to infect animals, although further research is needed. In stored oocysts, enzymes significantly reduced amylopectin content but not infectivity. Data shows that in fresh oocysts, amylopectin content, excystation, and infectivity are directly correlated 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 how 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 the infective sporozoites. Thus this research may add to further study of the role of amylopectin in infectivity of C. parvum, the ability to create a vaccine or control method based on amylopectin metabolism, and to increase detection and storage of oocysts.