Department or Program
Primary Wellesley Thesis Advisor
The intestinal parasite Cryptosporidium causes the diarrheal disease, cryptosporidiosis worldwide. Previous work done with Cryptosporidium parvum in the Ward lab showed that a C-type lectin glycoprotein known as CpClec is expressed on the surface of sporozoites and merozoites, the life cycle stages of Cryptosporidium which invades host epithelial cells in the human intestine. CpClec was also been found to be important for attachment and infection of host cells by binding to host expressed glycosaminoglycans. Loss or reduction of these glycosaminoglycans not only inhibited attachment but also infection. In order to further determine the function of CpClec, Jacob Ludington in the Ward lab developed two novel transgenic strains using CRISPR/Cas9 technology: one expressing a CpClec-HA tag (HA) and one with a CpClec gene deletion (KO). In both transgenic parasite strains, a nanoluciferase gene and a Neomycin resistance cassette was inserted that is expressed independently of CpClec. Using HCT-8 intestinal epithelial cells and CpClec expressing oocysts, it was confirmed that CpClec does play a role in infection in vitro by measurement of luciferase levels expressed by HA-tagged (HA) and deleted (KO) CpClec strains in infected HCT-8 cell lysates.
My role in this project was to participate in studies to determine the role of CpClec in an animal model in vivo and to determine its localization in the HA-tagged transgenic strain. Luciferase measurements in stool samples of infected mice showed that infection of interferon gamma (IFN-) deficient mice with CpClec KO oocysts resulted in significantly decreased infection compared to that with HA-tagged transgenic parasites in which the CpClec gene is intact. These results were confirmed through quantification of infection levels by analyzing 18S ribosomal RNA (rRNA) gene levels in stool samples via quantitative PCR. Additionally, immunofluorescence assays using an anti-HA antibody were used to determine that the localization of this protein throughout the Cryptosporidium life cycle appears to follow a specific pattern supporting its role in mediating infection. Confirmation of CpClec’s role in infection in in vivo models strengthens the importance of this protein as a potential target for drug or vaccine development against Cryptosporidium.
Available for download on Wednesday, April 20, 2022