Cryptosporidium (Crypto) causes diarrheal disease worldwide, which can be life-threatening in immunocompromised hosts such as untreated HIV/AIDS patients and malnourished children in resource- constrained countries. However, there is no consistently effective treatment for cryptosporidiosis in these vulnerable populations. Our long-term goal is to understand the role of Crypto proteins involved in mediating host cell attachment and invasion in order to develop novel interventions for cryptosporidiosis. We have identified and characterized Clec, a unique Crypto protein with three characteristics of proteins involved in attachment and invasion. Clec is 1) a glycosaminoglycan (GAG)-binding protein with 2) mucin-like and 3) C- type lectin domains. The objective of this application is to elucidate the role of Clec in Crypto infection. Clec is an extracellular type 1 membrane protein, which is conserved in C. parvum, C. hominis and C. muris, is expressed during infection in vitro, localizes to the apical surface and dense granules of invasive stages, binds to cell surface GAGs, specifically heparan sulfate proteoglycans (HSPGs) on intestinal epithelial cells and blocks C. parvum attachment to and invasion of these cells via interactions with these GAGs. These findings, strongly suggest that Clec plays a role in Crypto infection by mediating attachment and invasion and raise the possibility that it may serve as a target for intervention. However, until recently, it was not possible to determine the role of Crypto proteins using molecular genetics. Our central hypothesis is that Clec mediates Crypto attachment to and invasion of intestinal epithelial cells in vitro and promotes infection in vivo by binding to cell surface heparan sulfate proteoglycans. In this application, we propose to test this hypothesis in vitro and in vivo using CRISPR/Cas9 mediated gene-targeting approaches recently pioneered by our collaborator, Dr. Boris Striepen.
The Specific Aims are 1) to determine the role of C. parvum Clec (CpClec) in attachment to and invasion of human intestinal epithelial cells in vitro using gene-targeting approaches and 2) To determine the role of CpClec in infection in vivo using animal models of cryptosporidiosis. At the completion of these aims we expect to have established the role of CpClec in attachment and invasion in vitro and in infection in vivo using molecular genetics and complementary approaches. Future studies will be directed at determining the role of specific domains of Clec, and identifying and elucidating the roles of other GAG-binding Crypto proteins using gene-targeting approaches. The long-term goal is to determine whether CpClec and/or other GAG-binding proteins can serve as targets for drug or vaccine development.

Public Health Relevance

The intestinal parasite Cryptosporidium causes severe diarrhea in HIV/AIDS patients and malnourished young children for which treatment is unsatisfactory. The goal of this project is to determine the role of a Cryptosporidium protein, Clec, which is involved in infection by this parasite. The long-term goal is to develop effective treatment for this disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI127238-02
Application #
9302258
Study Section
AIDS-associated Opportunistic Infections and Cancer Study Section (AOIC)
Program Officer
Pesce, John T
Project Start
2016-06-22
Project End
2019-05-31
Budget Start
2017-06-01
Budget End
2019-05-31
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Tufts University
Department
Type
DUNS #
079532263
City
Boston
State
MA
Country
United States
Zip Code
02111