| Giardia intestinalis is an intestinal parasite that causes disease in humans and animals. The organism has two stages in its life cycle: trophozoite and cyst. The trophozoites grow and divide in the small intestines, and are able to become cysts by a process known as encystment. The cyst wall, which protects the parasite in the environment, is composed of protein and carbohydrate, with galactosamine as its major constituent. Galactosamine is not present in non-encysting trophozoites but is synthesized in encysting trophozoites through an inducible pathway of enzymes.;This study focuses on the regulation of these enzymes during the encystment process. Since encystment is vital for parasite transmission, investigation into how this pathway is regulated may lead to significant advances towards the development of chemotherapeutic agents to prevent further encystment. The genes for the enzymes in the pathway, glucosamine-6-phosphate isomerase, glucosamine 6-phosphate N-acetyltransferase, phosphoacetylglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-N-acetylglucosamine 4 ′-epimerase were each cloned, sequenced and expressed as GST-fusion proteins. The fusion proteins generated have the requisite enzymic activity, catalyzing the synthesis of UDP-GalNAc from fructose-6-phosphate. Furthermore, Northern and Western blot analyses indicated that both the mRNA and protein levels of these enzymes increase during encystment. Nuclear run-on assays showed that the increases were, at least in part, due to transcriptional activation. Immunofluorescence showed that isomerase is cytosolic, but that the last enzyme in the metabolic pathway, the epimerase, is localized near the developing cyst wall which suggests that the epimerase localizes to the cell membrane during encystment. Its activity may be coupled to a putative β-1,3 GalNAc transferase activity that polymerizes galactosamine into the cyst wall. Thus, these results show that the encystment pathway is turned on by transcriptional activation of all its genes, and that these genes may therefore be novel therapeutic targets for preventing further spread of giardiasis. |
