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. ... read moreGlycans 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-γ 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
- Component ID:
- tufts:20426
- To Cite:
- TARC Citation Guide EndNote
