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Albert Einstein College of Medicine
Characterization of the proteome of the Cryptosporidium parvum cyst outer wall
Tianmin Huang, Fa-Yun Che and the Albert Einstein Biodefense Parasite Proteomics
Introduction: Cryptosporidium parvum, a member of the Apicomplexa, infects epithelial
gastrointestinal cells of humans and other mammals. This pathogen has been respon-
sible for several large waterborne outbreaks of diarrheal disease resulting in significant
morbidity in immune competent hosts and mortality in immune compromised hosts.
Oocysts are environmentally resistant forms responsible for the transmission of this
organism in food and water. A defining characteristic of this life stage is the oocyst wall
a complex protective barrier consisting of a double layer of a protein-lipid-carbohydrate
matrix. The proteins of the oocyst spore wall are important potential targets.
Methods: Purified oocysts were excysted in taurocholate and oocyst walls isolated by
Percoll density gradient centrifugation as originally described by Arrowood and Streling
(J Parasitol. 73:314-319). Oocyst walls were solublized using different lysis buffers and
then separated using a 4-20% gradient SDS-PAGE gel and transferred to PVDF. As
expected this preparation reacted with a polyclonal rabbit anti-COWP1 we produced to
the peptides DKCAIHTVKV, PSGFVEEGNRC, and DHAGHGHGHGNQLLQEC. To
evaluate the glycosylation of oocyst wall proteins Glyko enzymatic deglycosylation kit
was used. Protein patterns obtained from 1D and 2D gels suggested that post-
translational modifications of oocyst wall proteins occurred. A 2D gel approach was
used for fractionation of the oocyst wall proteins prior to mass spectrometry analysis.
Oocyst walls were solubilized in a urea based lysis buffer (7.5M Urea, 2.5M thiourea,
40mM Tris (pH7.6), 2.5% octyl-β-glucoside, 6.25mM TCEP and proteinase inhibitor)
and the proteins separated by IEF (pH 3 to 11 IPG strip) followed by a 12% PAGE gel.
The gel was stained