O-Glycosylation and a Novel Bioengineered 3D Human Intestinal Tissue Model for Cryptosporidium
RePass, Maria.
2018
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Abstract: 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 in... read moreterventions 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 strains.
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 - ID:
- hh63t6972
- Component ID:
- tufts:25435
- To Cite:
- DCA Citation Guide EndNote
