Regulation of Carbohydrate Metabolism During <i>Giardia</i> Encystment<sup>1</sup>

Jarroll, Edward L.; Macechko, P. Timothy; Steimle, Paul A.; Bulik, Dorota A.; Karr, Craig; Keulen, Harry van; Paget, Timothy A.; Gerwig, Gerrit J.; Kamerling, J.P.; Vliegenthart, Johannes F.G.; Erlandsen, Stanley L.

doi:10.1111/j.1550-7408.2001.tb00412.x

Cited by 45 publications

(30 citation statements)

References 21 publications

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“…The cyst wall is composed of approximately 40% protein, and the remainder is carbohydrate (11). Because Giardia is considered one of the earliest eukaryotic cells, it occupies an enviable position on the scale of evolution of eukaryotic cells.…”

Section: Discussionmentioning

confidence: 99%

Oral Immunization of BALB/c Mice with Giardia duodenalis Recombinant Cyst Wall Protein Inhibits Shedding of Cysts

Larocque

Nakagaki²,

Lee

et al. 2003

Infect Immun

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The process of encystation is a key step in the Giardia duodenalis life cycle that allows this intestinal protozoan to survive between hosts during person-to-person, animal-to-person, waterborne, or food-borne transmission. The release of cysts from infected persons and animals is the main contributing factor to contamination of the environment. Genes coding for cyst wall proteins (CWPs), which could be used for developing a transmission-blocking vaccine, have been cloned. Since the immunogenicity of recombinant Giardia CWP is unknown, we have investigated the immunogenicity of recombinant CWP2 (rCWP2) and its efficacy in interfering with the phenomenon of encystation taking place in the small bowels of BALB/c mice vaccinated with the recombinant protein. Here we report that the immunization of BALB/c mice with rCWP2 stimulated the immune system in a manner comparable to that for a live infection with Giardia muris cysts. Fecal and serum anti-rCWP2 immunoglobulin A (IgA) antibodies were detected in the immunized mice. In addition, anti-rCWP2 IgG1 and IgG2a antibodies were detected in the serum. mRNAs coding for Th1 and Th2 types of cytokines were detected in spleen and Peyer's patch cells from immunized mice. When the vaccinated mice were challenged with live cysts, the animals shed fewer cysts. We conclude that rCWP2 is a possible candidate antigen for the development of a transmission-blocking vaccine.

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Section: Discussionmentioning

confidence: 99%

Oral Immunization of BALB/c Mice with Giardia duodenalis Recombinant Cyst Wall Protein Inhibits Shedding of Cysts

Larocque

Nakagaki²,

Lee

et al. 2003

Infect Immun

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“…The cyst wall is at least 40% protein, and the remainder is carbohydrate (14,15). The two known cyst wall proteins (CWPs) 1 contain similar domains, including a putative N-terminal signal peptide and five tandem, leucine-rich repeats (LRR) (16,17), and are reported to be phosphorylated (18).…”

mentioning

confidence: 99%

Mining the Giardia lamblia Genome for New Cyst Wall Proteins

Sun

McCaffery

Reiner

et al. 2003

Journal of Biological Chemistry

117

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The Giardia lamblia cyst wall (CW), which is required for survival outside the host and infection, is a primitive extracellular matrix. Because of the importance of the CW, we queried the Giardia Genome Project Database with the coding sequences of the only two known CW proteins, which are cysteine-rich and contain leucinerich repeats (LRRs). We identified five new LRR-containing proteins, of which only one (CWP3) is up-regulated during encystation and incorporated into the cyst wall. Sequence comparison with CWP1 and -2 revealed conservation within the LRRs and the 44-amino-acid N-flanking region, although CWP3 is more divergent. Interestingly, all 14 cysteine residues of CWP3 are positionally conserved with CWP1 and -2. During encystation, C-terminal epitope-tagged CWP3 was transported to the wall of water-resistant cysts via the novel regulated secretory pathway in encystation-secretory vesicles (ESVs). Deletion analysis revealed that the four LRRs are each essential to target CWP3 to the ESVs and cyst wall. In a deletion of the most C-terminal region, fewer ESVs were stained in encysting cells, and there was no staining in cysts. In contrast, deletion of the 44 amino acids between the signal sequence and the LRRs or the region just C-terminal to the LRRs only decreased the number of cells with CWP3 targeting to ESVs and cyst wall by ϳ50%. Our studies indicate that virtually every portion of the CWP3 protein is needed for efficient targeting to the regulated secretory pathway and incorporation into the cyst wall. Further, these data demonstrate the power of genomics in combination with rigorous functional analyses to verify annotation.

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“…All of these enzymes have been localized to the cytosol of encysting Giardia (Macechko et al, 1992). UDP-GalNAc is then converted into a unique b(1-3)-D-GalNAc homopolysaccharide by the action of a particle-associated b(1-3)GalNAc transferase, tentatively termed cyst wall synthase (CWS) (Jarroll et al, 2001). When trophozoites are induced to encyst, the first five of these enzymes are transcriptionally activated (Lopez et al, 2003) and the specific activities of all these enzymes increase (Macechko et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

Amino sugar phosphate levels in Giardia change during cyst wall formation

et al. 2004

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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.

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Regulation of Carbohydrate Metabolism During Giardia Encystment¹

Cited by 45 publications

References 21 publications

Oral Immunization of BALB/c Mice with Giardia duodenalis Recombinant Cyst Wall Protein Inhibits Shedding of Cysts

Oral Immunization of BALB/c Mice with Giardia duodenalis Recombinant Cyst Wall Protein Inhibits Shedding of Cysts

Mining the Giardia lamblia Genome for New Cyst Wall Proteins

Amino sugar phosphate levels in Giardia change during cyst wall formation

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Regulation of Carbohydrate Metabolism During Giardia Encystment1

Cited by 45 publications

References 21 publications

Oral Immunization of BALB/c Mice with Giardia duodenalis Recombinant Cyst Wall Protein Inhibits Shedding of Cysts

Oral Immunization of BALB/c Mice with Giardia duodenalis Recombinant Cyst Wall Protein Inhibits Shedding of Cysts

Mining the Giardia lamblia Genome for New Cyst Wall Proteins

Amino sugar phosphate levels in Giardia change during cyst wall formation

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Regulation of Carbohydrate Metabolism During Giardia Encystment¹