Harry van Keulen

Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14‐16 h. The intracellular phase lasted for 8‐10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4‐6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (‐15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.

Regulation of carbohydrate metabolism during Giardia encystment.

Abstract Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events related to the inducible synthesis of a novel N-acetyl-d-galactosamine (GalNAc) homopolymer, occur. Within the first 6 h of encystment, mRNA for glucosamine 6-P isomerase (GPI), the first inducible enzyme unique to this pathway appears, oxygen uptake rates double from non-encysting levels, and metronidazole (MTZ) inhibits oxygen uptake. Within 12 h, GPI and its activity are detectable and OU decreases 50% from non-encysting levels; glucoses stimulation and MTZs inhibition of oxygen uptake cease. In contrast, aspartate uptake remained constant throughout the 40 h monitored. Two genes, gpi 1 and 2 encode for GPI, but only gpi1 is expressed during encystment. Glucosamine 6-P (GlcN6P), the synthetic product of GPI, activates UDP-N-acetylglucosamine (UDP-GlcNAc) pyrophosphorylase, a downstream enzyme, 3 to 5-fold in the direction of UDP-GlcNAc synthesis. UDP-GlcNAc is epimerized to UDP-GalNAc and UDP-GalNAc is polymerized by “cyst wall synthase” (β 1 ;ra 3 GalNAc transferase) into a highly insoluble β 1,3-linked homopolymer. This GalNAc polysaccharide, the major component of cyst wall filaments, forms, in conjunction with polypeptides, the outer cyst wall of Giardia.

A three nucleotide signature sequence in small subunit rRNA divides human Giardia in two different genotypes.

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.

Transcription regulation is demonstrated for five key enzymes in Giardia intestinalis cyst wall polysaccharide biosynthesis

The cyst wall of Giardia intestinalis contains proteins and a novel N-acetylgalactosamine (GalNAc) polysaccharide, which is its major constituent. GalNAc is not present in growing trophozoites, but is synthesized during encystment via an inducible pathway of enzymes that produce UDP-GalNAc from fructose 6-phosphate. This report focuses on the regulation of these enzymes and thus the genes for glucosamine 6-phosphate N-acetyltransferase (GNA), phosphoacetylglucosamine mutase (AGM), UDP-N-acetylglucosamine pyrophosphorylase (UAP), and UDP-N-acetylglucosamine 4-epimerase (UAE) were cloned and expressed in Escherichia coli. Each of these expressed enzymes had the predicted activity and was used to generate antibodies. Northern and Western blot analyses demonstrated that both the mRNA and protein levels for all of these enzymes increase during encystment. Nuclear run-on assays of these and the previously analyzed glucosamine 6-phosphate deaminase (GNP; glucosamine 6-P isomerase) showed that all of the genes responsible for UDP-GalNAc synthesis during encystment are induced at the transcription level.

Cloning of Two Putative Giardia lamblia Glucosamine 6‐Phosphate Isomerase Genes Only One of Which is Transcriptionally Activated During Encystment

ABSTRACT The biosynthesis of the carbohydrate component of the cyst wall of the protozoan parasite Giardia lamblia, a polymer of N‐acetylgalactosamine (GalNac). is by a pathway that is initiated with the conversion of fructose 6‐phosphate to glucosamine 6‐phosphate by an aminating isomerase. glucose 6‐phosphate isomerase. This enzyme appears only after Giardia trophozoites an: induced to start the production of cyst wall components after bile is added. To investigate whether induction of glucosamine 6‐phosphate isomerase is by protein modification or by transcription activation, its gene was cloned and sequenced. Two genes, gpi1 and gpi2, encoding putative glucosamine 6‐phosphate isomerases were identified but only one, gpi1 was expressed. The transcript for gpi1 appeared not earlier than 6 h after cells were induced with bile salts. These results show that the first enzyme in the pathway leading to GalNac synthesis in encysting Giardia cyst wall biosynthesis is under transcriptional control.

Giardia sp.: comparison of electrophoretic karyotypes.

Species in the genus Giardia have been named on the basis of host specificity, cell dimensions, and median body morphology. Despite these criteria, the species taxonomy of Giardia is still in question. To investigate Giardia taxonomy on a molecular level, Giardia chromosomal DNA was analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE) and transverse alternating field electrophoresis (TAFE). Chromosomal DNA of G. duodenalis isolates (human, muskrat, sheep, dog, beaver), G. muris (mouse), and G. ardeae (great blue heron) were subjected to OFAGE and TAFE analyses. Comparable DNA patterns were obtained by both electrophoretic methods, but OFAGE required 8 days while TAFE required only 3 days. DNA patterns among all G. duodenalis isolates, although quite similar to each other, were distinctly different from those of G. muris and G. ardeae; G. muris and G. ardeae DNA patterns were distinctly different from each other. A G. duodenalis (Portland 1) total DNA probe hybridized to the DNA of all G. duodenalis isolates on Southern blots, but not detectably to G. muris and G. ardeae DNA. Similarly, G. muris and G. ardeae total DNA probes only hybridized detectably to their respective DNA. One probe that appears to hybridize to the DNA of all G. duodenalis and to G. ardeae DNA rather than G. muris DNA has been developed. Another probe that hybridizes only to G. muris and G. ardeae DNA has been developed. These data suggest that the differentiation of Giardia isolated from host and environmental samples may eventually be accomplished by DNA probes. Additionally, these techniques perhaps combined with other criteria may lead to the establishment of a sound taxonomic scheme for this genus.

Yeasts present during spontaneous fermentation of Lake Erie Chardonnay, Pinot Gris and Riesling

The composition of wine yeast populations, present during spontaneous fermentation of Chardonnay, Pinot Gris and Riesling from the Lake Erie Region was studied. A combination of biochemical and molecular techniques was used to identify non-Saccharomyces and Saccharomyces yeast isolates. The biochemical techniques included analysis of yeast isolates by sugar fermentation and carbon and nitrogen assimilation. Molecular techniques involved ribotyping of a highly variable segment in the 26S rRNA gene using DNA sequence analysis and restriction fragment length polymorphism of amplified DNA. The results show that of the non-Saccharomyces yeasts, several related species of Hanseniaspora, were the most abundant yeasts present during early stages of fermentation. Later in fermentation S. cerevisiae dominated, and based on biochemical analyses consisted of a heterogeneous group of genotypes. There were no major differences in yeast populations among the three types of juice analyzed.

Differential expression of proteins induced by lead in the Dwarf Sunflower Helianthus annuus.

The Dwarf Sunflower (Helianthus annuus) is a hyperaccumulator of toxic metals including cadmium (Cd), mercury (Hg), nickel (Ni), and lead (Pb). In order to identify stress response to Pb, plants were exposed to a mixture of 30 mg/l of three ions, Cd, Cr, and Ni, with and without Pb. Soluble proteins were resolved by two-dimensional gel electrophoresis. Four proteins were differentially expressed and very abundant in the leaf samples after plants were exposed to all these four metals. The first protein spot contained two proteins: chitinase and a chloroplast drought-induced stress protein CDSP-34. The second spot contained a thaumatin-like protein. Two proteins in spot 3 were identified as heat-shock cognate 70-1 and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Several peptides were identified in spot 4 but none could be matched to any sequence in the NCBI database.

Giardia intestinalis Glucosamine 6‐Phosphate Isomerase: the Key Enzyme to Encystment Appears to be Controlled by Ubiquitin Attachment

Abstract The cyst wall of the parasitic protozoan, Giardia intestinalis, is composed of a polymer of N-acetylgalactosamine, the precursor of which is synthesized by an inducible enzyme pathway. The first enzyme in this pathway, glucosamine 6-phosphate isomerase, is transcriptionally regulated. During encystment and in mature cysts this isomerase appears to be modified by ubiquitin attachment. Thus, it might be targeted for destruction by an ubiquitin-mediated pathway, suggesting that glucosamine 6-phosphate isomerase expression is tightly regulated.

The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris.

SummaryThe total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined. The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp. The spacer contains long direct repeats and is heterogeneous in size. The LSU-rRNA of G. muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli. The LSU-rRNA has a size comparable to the 23S rRNA of E. coli but shows structural features typical for eukaryotes. Some variable regions are typically small and account for the overall smaller size of this rRNA. The structure of the G. muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.