During encystment, Giardia trophozoites become encased in a filamentous extracellular matrix of their own making that consists of novel cyst wall proteins (Cwp) 1, 2 and 3, and a novel 2-acetamido-2-deoxy-d-galactan we are naming giardan. Giardan is synthesized from glucose via sugar phosphate intermediates to UDP-GalNAc by inducible, cytosolic enzymes. The UDP-GalNAc is fixed into giardan apparently by an inducible, particle-associated transferase. Regulation of this synthesis appears to centre around pyrophosphorylase, epimerase and cyst wall synthase (Cws) activities. Pyrophosphorylase seems to be involved in making sufficient UDP-N-acetylglucosamine (GlcNAc) to drive the epimerase kinetics toward UDP-GalNAc synthesis, while the Cws removes intracellular UDP-GalNAc, extruding it as giardan and thus preventing an increased intracellular concentration of UDP-GalNAc that could drive the reaction toward GlcNAc synthesis. Cyst wall proteins have been localized to encystment-specific vesicles (ESVs), but whether or not this is true for giardan is unknown. Also unknown is whether or not the cyst wall proteins and giardan are covalently linked. It remains unknown how or whether Giardia degrades giardan during excystation.
The pyrimidine metabolism of Tritrichomonas foetus (KV 1) was studied using whole cells and cell homogenates. Pyrimidines and pyrimidine nucleosides were readily incorporated into nucleic acids. Orotate and aspartate were not incorporated into pyrimidine bases. Enzymes of the pyrimidine salvage pathway (i.e., thymidine and uridine phosphorylases and uridine kinase) were detected in trophozoite homogenates, but the activities of de novo pyrimidine synthesis enzymes (i.e., carbamoylphosphate synthase, aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase) were below the level of detection in these same homogenates. The evidence presented supports the proposal that T. foetus is incapable of synthesizing pyrimidines de novo but is capable of salvaging preformed pyrimidines and pyrimidine nucleosides from the growth medium and that enzymes of this parasite's pyrimidine salvage pathway are not organelle-associated.
ABSTRACT. The nucleotide sequence of the 16S rRNA gene, part of the 23S rRNA gene and the spacer DNA region was determined for Giardia duodenalis , obtained from humans in The Netherlands (AMC‐4) and Washington State (CM). These rDNA sequences differ from other G. duodenalis isolates (Portland‐1 and BRIS/83/HEPU/106) both of which have virtually identical rDNA sequences. The most characteristic feature was found close to the 5’end of the 16S rRNA. The Portland‐1 ‐ Bris/83/HEPU/106 type has GCG in position 22–24, while AMC‐4 and CM have AUC in this position. These two sequences, present in an otherwise conserved region of the 16S rRNA, are “signature” sequences, which divide Giardia isolates into two different groups.
Trophozoites of Giardia ardeae were obtained from the great blue heron (Ardea herodias) and established in axenic culture using the TYI-S-33 medium. The generation time in culture for G. ardeae was 22-25 hr, which was 3-fold longer than for Giardia duodenalis (WB strain). A morphological comparison of trophozoites in the original intestinal isolate to those grown in culture revealed that they were identical for the following characteristics: a pyriform-shaped body, a ventral adhesive disc with a deep notch in the posterior border, teardrop-shaped nuclei, pleomorphism in median body structure ranging from a round-oval appearance (Giardia muris type) to that of a clawhammer (G. duodenalis type), and a single caudal flagellum on the right side (as viewed dorsally) with the left one being rudimentary. Analysis of the chromosomal migration patterns was performed by orthogonal-field-alternation gel electrophoresis and demonstrated that the pattern for G. ardeae was distinctly different from that for G. duodenalis (Portland 1-CCW strain). Bacterial symbionts were seen attached to trophozoites in the original isolate but could not be detected in cultured trophozoites using scanning electron microscopy, fluorescence light microscopy using the Hoechst 33258 dye for DNA localization, or by standard microbiological techniques using nonselective media for growing aerobic or anaerobic bacteria. This study demonstrated that avian-derived Giardia could be grown in axenic culture; based on morphological criteria and chromosomal migration patterns, that G. ardeae should be considered a distinct species; and that rationale for determining Giardia spp., based on median body structure alone, should no longer be considered adequate for classification at the species level.
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 V max and a smaller K m 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.
Abstract In this review, we examine the state-of-the-art technologies (gas and liquid chromatography, mass spectroscopy and nuclear magnetic resonance, etc.) in the well-established area of metabolomics especially as they relate to protozoan parasites.
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