The overall goals of this project are to elucidate the terminal steps of encystation by Giardia lamblia, and the regulation of chitin synthetaase (CS), a key enzyme in cyst wall biosynthesis. This is a basic problem in differentiation and relevant to transmission of an enteric disease of particular importance in children. These goals are feasible because we have now: 1) devised an encystation medium that induces large number of G lamblia cysts in vitro and (2) demonstrated developmentally regulated CS in G. lamblia and begun to characterize and compare it to the CS of Entamoeba invadens. Now we propose to: 1. Define the influence of selected parasite and host factors on the terminal steps of G. lamblia encystation, in order to elucidate the control of differentiation and maximize the yield of biologically active cysts. 2. Compare in vitro derived cysts to cysts from natural infections by the following criteria: morphology, ability to excyst, and infectivity for suckling mice. 3. Elucidate the developmental regulation of chitin synthetase (CS) activities of G. lamblia and Entamoeba invadens by studies designed to assess the effects of encystation stimuli on CS activity. 4. Purify and characterize the CS from both parasites in order to determine their cellular locations, products, and properties. 5. Screen expression DNA libraries with antibodies and gene probes and for CS activity in order to isolate the CS structural genes, determine their DNA sequences and deduce the amino acid sequences of their gene products. 6. Prepare probes for the cloned genes and study their regulation. These probes will be used to determine the effects of encystation stimuli on CS gene expression at the transcriptional level. The results of this proposal should yield new insights into factors that control the life cycle of an important pathogen, as well as provide unlimited supplies of pure G. lamblia cysts for basic and applied research, and help define the roles of CS in other medically and economically important organisms.
