The role of a novel protozoan C-type lectin in Cryptosporidium-host cell interactions.
Ludington, Jacob.
2016
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Abstract: Cryptosporidium is an environmentally-resistant apicomplexan parasite that causes significant diarrheal disease worldwide. In spite of the devastating global impact of cryptosporidiosis, there is no vaccine or effective therapy to mitigate disease in vulnerable populations, in part because the molecular mechanisms underlying Cryptosporidium-host cell interactions are poorly understood. G... read morelycans and glycan binding proteins have been demonstrated to play a major role during infection, and Cryptosporidium encodes at least thirty mucin-like domain-containing proteins, some of which are implicated in mediating attachment to and invasion of host cells. In this thesis, we identify and characterize C. parvum Clec (CpClec), a novel mucin-like domain-containing protein that additionally contains a C-type lectin domain (CTLD) with orthologs in all gut invading, cyst-forming apicomplexans whose genomes have been sequenced and annotated. The C-type lectin domain is traditionally a Ca2+-dependent glycan-binding domain that mediates cell-cell interactions in a wide range of organisms from humans to viruses. However, this is the first report of a C-type lectin domain in a protozoan. CpClec is predicted to be a type 1 membrane protein, with a CTLD, an O-glycosylated, mucin-like domain, N-glycosylation sites, a transmembrane domain, and a cytoplasmic tail containing multiple tyrosine-based signaling or sorting motifs. CpClec displays several characteristics of canonical CTLD-containing proteins and contains motifs associated with Ca2+-dependent glycan binding. Expression is developmentally regulated and its native size is significantly larger than predicted, suggesting that CpClec may be modified by O-glycosylation. The finding that CpClec localized to the apical region of invasive stages similar to other mucin-like domain-containing glycoproteins prompted us to further investigate its role during Cryptosporidium infection using an Fc-tagged recombinant protein. CpClec-Fc displayed Ca2+-dependent, saturable binding to HCT-8 and Caco-2 intestinal epithelial cell lines and competitively inhibited C. parvum attachment to and infection of HCT-8 cells. Binding of CpClec-Fc to HCT-8 and Caco-2 cells was specifically inhibited by sulfated glycosaminoglycans, particularly heparin and heparan sulfate, and binding was reduced after disruption of surface glycosaminoglycans on HCT-8 cells. Similar to Cplec-Fc, C. parvum attachment to and infection of HCT-8 were inhibited by glycosaminoglycans and were reduced after disruption of cell surface glycosaminoglycans. Lastly, CpClec-Fc binding and C. parvum sporozoite attachment were significantly decreased in CHO cell mutants defective in glycosaminoglycan synthesis. In order to directly investigate the role of CpClec during infection, we generated transgenic C. parvum strains in which the Clec locus was deleted or fused to an HA-epitope tag. CpClec-HA was detected in specific C. parvum intracellular stages and localized to a unipolar region beneath the parasitophorous vacuolar membrane. Knockout of CpClec inhibited C. parvum infection of HCT-8 cells and significantly reduced oocyst shedding in IFN- deficient mice. Together, these results indicate that CpClec is a novel C-type lectin that mediates C. parvum attachment and infection via Ca2+-dependent binding to sulfated proteoglycans on intestinal epithelial cells.
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Immunology.
Advisors: Honorine Ward, and Mercio Perrin.
Committee: John Leong, Joan Mecsas, and Miguel Stadecker.
Keywords: Parasitology, Molecular biology, and Biochemistry.read less - ID:
- j098zp72s
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