OKHUYSEN PC, PADDA RS, TSAI A, CHAPPELL CL; Interscience Conference on Antimicrobial Agents and Chemotherapy (41st : 2001 : Chicago, Ill.).
Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 2001 Dec 16-19; 41: abstract no. P-1133.
The University of Texas Houston Medical School and School of Public Health, Houston, TX
BACKGROUND: C. parvum can cause diarrhea in healthy individuals and in patients with AIDS. C. parvum proteases have been associated with the release of sporozoites from oocysts and their specific inhibition blocks excystation in vitro. Aminopeptidases have been implicated in the processing of parasite adhesion molecules on the sporozoite surface. Since C. parvum cannot be cultured in vitro, we sought to characterize and express a recombinant version of this putative virulence factor. METHODS: The entire cpapn gene was identified and cloned by screening a large insert P1 artificial chromosome library with a probe identified from a C. parvum genome survey sequencing project .cpapn was then subcloned into an expression vector. Reverse transcription polymerase chain reaction (RT-PCR) was used to confirm in vivo expression of cpapn during infection of temperature sensitive fetal human intestinal enterocytes in culture. Amplification of the human glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as a control. RESULTS: Analysis of the predicted protein encoded by the 2.3 kb gene demonstrated a high degree of homology with prokaryotic as well as eukaryotic aminopeptidases with a predicted Mr of ~89,000. The active site sequence is highly conserved when compared to other Apicomplexan aminopeptidases such as Plasmodium. A single RGD-containing motif was identified. The recombinant protein was approximately 90kDa in size. RT PCR confirmed the expression of cpapn during the early stages of the parasitic infection in infected cells but not in uninfected cells. CONCLUSIONS: 1) C. parvum aminopeptidase N has been sequenced cloned and expressed in vitro. 2) Unlike other proteins in this class, the adhesive amino acid motif RGD was identified. 3) Aminopeptidase N RT PCR amplification products are detectable in the early stages of parasite infection.
Publication Types:
Keywords:
- Animals
- Antigens, CD13
- Base Sequence
- Cloning, Molecular
- Cryptosporidium parvum
- DNA, Complementary
- Gene Expression
- Gene Library
- Humans
- In Vitro
- Oocysts
- Recombinant Proteins
- Reverse Transcriptase Polymerase Chain Reaction
- Sporozoites
- analysis
- genetics
- immunology
Other ID:
UI: 102270115
From Meeting Abstracts