The parasite Giardia intestinalis exists as a trophozoite (vegetative) that infects the human small intestine, and a cyst (infective) that is shed in host faeces. Cyst viability in the environment depends upon a protective cyst wall, which consists of proteins and a unique b(1-3) GalNAc homopolymer. UDP-GalNAc, the precursor for this polysaccharide, is synthesized from glucose by an enzyme pathway that involves amino sugar phosphate intermediates. Using a novel method of microanalysis by capillary electrophoresis, the levels of amino sugar phosphate intermediates in trophozoites before encystment, during a period of active encystment and after the peak of encystment were measured. These levels were used to deduce metabolic control of amino sugar phosphates associated with encystment. Levels of amino sugar phosphate intermediates increased during encystment, and then decreased to nearly non-encysting levels. The most pronounced increase was in glucosamine 6-phosphate, which is the first substrate unique in this pathway, and which is the positive effector for the pathway's putative rate-controlling enzyme, UDP-GlcNAc pyrophosphorylase. Moreover, more UDP-GalNAc than UDP-GlcNAc, its direct precursor, was detected at 24 h. It is postulated that the enhanced UDP-GalNAc is a result of enhanced synthesis of UDP-GlcNAc by the pyrophosphorylase, and its preferential conversion to UDP-GalNAc. These results suggest that kinetics of amino sugar phosphate synthesis in encysting Giardia favours the direction that supports cyst wall synthesis. The enzymes involved in synthesis of UDP-GalNAc and its conversion to cyst wall might be potential targets for therapeutic inhibitors of Giardia infection.
ABSTRACT. The protozoan parasite Giardia intestinalis has a simple life cycle consisting of an intestinal trophozoite stage and an environmentally resistant cyst stage. The cyst is formed when a trophozoite encases itself within an external filamentous covering, the cyst wall, which is crucial to the cyst's survival outside of the host. The filaments in the cyst wall consist mainly of a β (1–3) polymer of N‐acetylgalactosamine. Its precursor, UDP‐N‐acetylgalactosamine, is synthesized from fructose 6‐phosphate by a pathway of five inducible enzymes. The fifth, UDP‐N‐acetylglucosamine 4′‐epimerase, epimerizes UDP‐N‐acetylglucosamine to UDP‐N‐acetylgalactosamine reversibly. The epimerase of G. intestinalis lacks UDP‐glucose/UDP‐galactose 4′‐epimerase activity and shows characteristic amino acyl residues to allow binding of only the larger UDP‐N‐acetylhexosamines. While the Giardia epimerase catalyzes the reversible epimerization of UDP‐N‐acetylglucosamine to UDP‐N‐acetylgalactosamine, the reverse reaction apparently is favored. The enzyme has a higher Vmax and a smaller Km in this direction. Therefore, an excess of UDP‐N‐acetylglucosamine is required to drive the reaction towards the synthesis of UDP‐N‐acetylgalactosamine, when it is needed for cyst wall formation. This forms the ultimate regulatory step in cyst wall biosynthesis.
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