O-Glycosylation and a Novel Bioengineered 3D Human Intestinal Tissue Model for Cryptosporidium
Cryptosporidium spp. are the causative agents of diarrheal disease worldwide, with the
greatest burden on the immune-compromised and malnourished young children in resource
poor countries. In vitro culture models to study this parasite with physiological
relevance to in vivo infection remain sub-optimal. Thus, the pathogenesis of
cryptosporidiosis remains poorly characterized, and i... read morenterventions for the disease are
limited. The lack of effective treatments makes the study of this organism and the
development of new interventions of the utmost importance. Cryptosporidium employs
mucin-like glycoproteins to attach to and infect host intestinal epithelial cells.
O-glycans, specifically the Tn antigen (GalNAc 1-Ser/Thr) on these glycoproteins
have been shown to be essential for these processes as a Tn-specific antibody and
Tn-specific lectins block attachment and irreversibly inhibit infection. However, the
enzymes catalyzing their synthesis have not been studied. Previously, we identified four
genes encoding putative polypeptide N-acetylgalactosaminyl transferases (ppGalNAc-Ts) in
the genomes of three Cryptosporidium spp. My project focused on the in silico analysis,
cloning, expression, purification and characterization of one of the four ppGalNAc-Ts
within C. parvum, Cp-ppGalNAc-T4. This enzyme contains the characteristic domains and
motifs conserved in ppGalNAc-Ts family enzymes and is expressed at multiple time points
during in vitro infection. Soluble, recombinant Cp-ppGalNAc-T4 functions primarily as an
"initiating" enzyme with a strong preference for UDP-GalNAc over other nucleotide sugar
donors. Given the importance of mucin type-O-glycosylation within Cryptosporidium spp.,
the enzymes that catalyze their synthesis may serve as potential therapeutic targets.
While characterizing mucin-like glycoproteins and enzymes that glycosylate them is
important, the lack of robust in vitro culture systems remains a serious impediment to
fully understanding their function and hinders screening of potential interventions
which target them. We evaluated the potential of a novel bioengineered three-dimensional
(3D) human intestinal tissue model to support long-term infection by C. parvum. We found
that C. parvum infected and developed in this model for at least 17 days. Contents from
infected 3D tissue models could be transferred to fresh 3D tissue models to establish
new infections for at least three rounds. Asexual and sexual stages and the formation of
new oocysts were observed during the course of infection. We further improved the model
by using human intestinal stem-cell derived "enteroids" to replace the transformed cell
lines. Ultimately, a 3D model system capable of supporting continuous Cryptosporidium
infection will be a useful tool for further advancing the study of host-parasite
interactions, identification of putative drug targets, screening of potential
interventions, and propagation of wild type and genetically modified Cryptosporidium
Thesis (Ph.D.)--Tufts University, 2018.
Submitted to the Dept. of Immunology.
Advisors: Honorine Ward, and Mercio PereiraPerrin.
Committee: Stephen Bunnell, Noorjahan Panjwani, Ananda Roy, and Marc-Jan Gubbels.
Keywords: Parasitology, Biochemistry, and Biomedical engineering.read less