Giardia lamblia, which is spread by the fecal-oral route, is an important parasitic cause of diarrhea in the US and the developing world. The infectious and diagnostic stage of Giardia is the quadranucleate cyst, which has a wall composed in part by fibrils of a unique GalNAc homopolymer. During the previous funding period, we have made many discoveries concerning glycosylation of Giardia proteins (Aim 1) and the formation of the cyst wall (Aim 2). These discoveries have suggested two related hypotheses that will be pursued in the proposed experiments. First, protein glycosylation in Giardia is much simpler than that of the human host or of model eukaryotes such as Saccharomyces. Therefore studies of Giardia glycans in Aim 1 will provide insights concerning the diversity and evolution of protein glycosylation, as well as providing a better description of Giardia glycoproteins. The second hypothesis is that the cyst wall of Giardia is composed of a small number of proteins, which are lectins that bind fibrils of the GalNAc homopolymer. Studies in Aim 2 that characterize enzymes involved in synthesis and degradation of the Giardia cyst wall will lead to insights into the mechanism of pathogenesis of this parasite and provide an alternative wall model that is distinct from those of fungi that contain multiple sugar polymers and ~100 proteins. Progress towards Aim 1 included demonstration that Giardia makes a 2-sugar N-glycan precursor rather than the 14-sugar precursor made by metazoans and fungi. The Giardia oligosaccharyl transferase (OST) that transfers N-glycans has a single subunit rather than 8 subunits. There is selection for sites of N- linked glycans in eukaryotes with N-glycan-dependent quality control (QC) of protein folding, but there is no such selection in Giardia.UDP-GlcNAc is the only nucleotide-sugar transported into the ER lumen of Giardia. Proposed experiments towards Specific Aim 1 will characterize the single-subunit OST of Giardia and characterize what we predict is a unique GPI anchor of Giardia. Progress toward Aim 2 included use of wheat germ agglutinin, which binds to the Giardia N-glycans, to dramatically enrich secreted and membrane proteins. Cyst-specific glycoproteins included a candidate for the GalNAc homopolymer synthase. The GalNAc homopolymer, which was isolated free of protein, maintains its hollow spherical shape based upon interlocking loops of curled fibrils. The Leu-rich repeat domain of the Giardia cyst wall protein 1 is a lectin that binds the GalNAc homopolymer. Proposed experiments towards Specific Aim 1 will explore further a curled fibril and lectin model of the Giardia cyst wall, isolate the synthase for the unique GalNAc homopolymer, and characterize the glycohydrolase that breaks down the GalNAc polymer during excystation.

Public Health Relevance

Giardia lamblia, which is spread by poor hygiene, is an important cause of diarrhea in the US, Europe, and developing countries. Studies of the sugars that cover the parasite and that make its protective cyst wall are important to understand how Giardia causes disease and may in the long term lead to better diagnostics, vaccines, or therapeutics.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AI048082-09
Application #
8659333
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mcgugan, Glen C
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Boston University
Department
Biochemistry
Type
Schools of Dentistry/Oral Hygn
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02118
Samuelson, John; Robbins, Phillips W (2015) Effects of N-glycan precursor length diversity on quality control of protein folding and on protein glycosylation. Semin Cell Dev Biol 41:121-8
Bushkin, G Guy; Motari, Edwin; Carpentieri, Andrea et al. (2013) Evidence for a structural role for acid-fast lipids in oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. MBio 4:e00387-13
Samuelson, John; Robbins, Phillips (2011) A simple fibril and lectin model for cyst walls of Entamoeba and perhaps Giardia. Trends Parasitol 27:17-22
Chatterjee, Aparajita; Carpentieri, Andrea; Ratner, Daniel M et al. (2010) Giardia cyst wall protein 1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. PLoS Pathog 6:e1001059
Bushkin, G Guy; Ratner, Daniel M; Cui, Jike et al. (2010) Suggestive evidence for Darwinian Selection against asparagine-linked glycans of Plasmodium falciparum and Toxoplasma gondii. Eukaryot Cell 9:228-41
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Cui, Jike; Das, Suchismita; Smith, Temple F et al. (2010) Trichomonas transmembrane cyclases result from massive gene duplication and concomitant development of pseudogenes. PLoS Negl Trop Dis 4:e782
Banerjee, Sulagna; Robbins, Phillips W; Samuelson, John (2009) Molecular characterization of nucleocytosolic O-GlcNAc transferases of Giardia lamblia and Cryptosporidium parvum. Glycobiology 19:331-6
Cui, Jike; Smith, Temple; Robbins, Phillips W et al. (2009) Darwinian selection for sites of Asn-linked glycosylation in phylogenetically disparate eukaryotes and viruses. Proc Natl Acad Sci U S A 106:13421-6
Pal, Dibyarupa; Banerjee, Sulagna; Cui, Jike et al. (2009) Giardia, Entamoeba, and Trichomonas enzymes activate metronidazole (nitroreductases) and inactivate metronidazole (nitroimidazole reductases). Antimicrob Agents Chemother 53:458-64

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