Amino sugar phosphate levels in Giardia change during cyst wall formation

Abstract

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.

Sphingolipids, Lipid Rafts, and Giardial Encystation: The Show Must Go On

“…Giardial cysts have tetranucleate-containing basal bodies and axonemes, which are close in proximity to nuclei and fragmented discs [15]. The cyst walls are made of proteins and β-(1,3)-N-acetylgalactosamine homopolymer, which provides a solid support to the cyst wall [16–19]. …”

Current Therapeutics, Their Problems, and Sulfur-Containing-Amino-Acid Metabolism as a Novel Target against Infections by “Amitochondriate” Protozoan Parasites

“…Potential targets for the development of antiamoebic drugs include the glycolytic pathway (namely, pyrophosphate-dependent phosphofructokinase and pyruvate kinase) (103,200), alcohol dehydrogenase 2 (90, 337), cysteine proteases (157,252), isoprenyltransferases (170,195), and sulfur-containing-aminoacid metabolism (233). For the development of new chemotherapeutics against giardiasis, several candidates have been investigated, including guanine phosphoribosyltransferase, a key enzyme in the purine salvage pathway (222), and biosynthesis of a novel ␤-(1,3)-N-acetyl-D-galactosamine homopolymer [including 4Ј-epimerase and ␤-(1,3)-N-acetyl-D-galactosamine transferase] (102,148,285). Encystation and excystation are unique cellular processes that fulfill the criteria for rational drug targets.…”

Enzymatic analysis of uridine diphosphate N-acetyl-d -glucosamine

“…Capillary electrophoresis showed excellent separation of UDP-GlcNAc and UDPGalNAc in a method that required at least 18 min [11]. A similar capillary zone electrophoresis method was used to resolve the sugar nucleotides from Giardia intestinalis [12]. While each of these methods simultaneously determines many sugar nucleotide concentrations, they are limited by low-throughput, difficulty resolving sugar nucleotide epimers or high fixed costs.…”