Cryptosporidium is a ubiquitous waterborne
parasite of worldwide distribution. It is currently considered
a pathogen of significant medical importance because of its
ability to cause a chronic, severe and frequently fatal diarrhea
in immunocompromised patients such as those with AIDS. To
date, there is no consistently effective treatment for Cryptosporidium
spp. infection. Our work has focused on identifying and characterizing
the parasite molecules involved in adhesion and invasion of
C. parvum invasive stages (zoites) into
the intestinal epithelial cell with the long-term goal of
developing interventions to interrupt these processes in order
to prevent and treat infection. Previous studies have shown
that in Cryptosporidium, unique mucin-like glycoproteins,
and specifically the glycotopes on these antigens, are important
for entry of the parasite into gut epithelial cells. The C.
parvum genome, recently sequenced and annotated, contains
more that 30 ORFs encoding putative mucins; to date, only
three of these have been characterized. We have identified
6 mucins, clustered together on a single locus, that are expressed
during intracellular development. Our hypothesis is that
these mucins play a role in the processes of attachment and
invasion of C. parvum into epithelial cells. This hypothesis
will be tested by the completion of two specific aims:
Specific aim 1: To determine expression
and localization of the mucins in C. parvum invasive
and intracellular stages.
We will express each mucin in E. coli, and use the
recombinant proteins to generate polyclonal antibodies. These
will be used in immunofluorescence assays and Western blots
to determine localization and expression of the native proteins.
Specific aim 2: To evaluate the ability
of anti-mucin antibodies to block C. parvum infection
in vitro.
Polyclonal antibodies will be generated against recombinant
C. parvum glycoproteins expressed in Toxoplasma
gondii. These antibodies, and the antibodies generated
against E. coli recombinants, will be tested for
their ability to block Cryptosporidium infection
of intestinal epithelial cells in vitro.
At the completion of this pilot project we will have data
on localization, expression, post-translational modification
and function of 6 previously uncharacterized cryptosporidial
mucins. This information will improve our understanding of
the processes that allow C. parvum to invade and
develop in host epithelial cells as well as identifying potential
targets for the design of effective treatments and vaccines
for this parasite.
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