The broad, long-term objective of the present research proposal is to use biochemical approaches to identify a potential target enzyme in Cryptosporidium parvum for anti-cryptosporidiosis chemotherapy, and to rely on molecular biological techniques to provide enough of such enzyme for thorough characterizations and specific inhibitor designs. All protozoan parasites studied to-date lack the capability of de novo synthesis of purine nucleotides. A close relative of C. parvum, Eimeria tenella, is known to rely primarily on the activity of one purine salvage enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) for survival. IT is highly likely that C. parvum is also deficient in de novo purine nucleotide synthesis, and has to depend on an HGXPRTase as the primary purine salvage enzyme for survival. These two assumptions will be first verified by: 1) incorporation of radiolabeled precursors into the nucleic acids of C. parvum schizonts growing in cell cultures; 2) pulse-chase experiments with radiolabeled precursors on C. parvum sporozoites, followed by high performance liquid chromatographic (HPLC) analysis of the labeled purine nucleotide pool; 3) assays of enzyme activities involved in purine metabolism in cell-free extracts of C. parvum oocysts. A map of major purine salvage pathways can be concluded from these studies, which will, in turn, help to pinpoint the crucial purine salvage enzyme in C. parvum. The enzyme will be purified from C. parvum oocysts, partially characterized and partly sequenced from its N-terminus. A cloned full-length cDNA encoding this enzyme will be identified in a C. parvum cDNA library. Probes to be used for this purpose will be derived from polymerase chain reaction (PCR) products synthesized from the primers by the N-terminal peptide sequence or the consensus peptide sequences shared by all the known purine phosphoribosytransgerases in eukaryotes. Alternatively, cDNAs encoding mouse HGPRTase or Schistosoma mansoni HGPRTase or the antibodies to the purified C. parvum enzyme can be used to screen the library. The identified cDNA will be subcloned, sequenced and ligated into a phoA- promoter-containing bacterial plasmid pBAce for expression in transformed Escherichia coli (which expressed S. mansoni HGPRTase in its native form up to 60% of the total E. Coli protein in our laboratory). The purified recombinant enzyme will then subject to indepth kinetic analyses, crystallization for X-ray diffraction analysis of three-dimensional structure and computer graphic modeling for specific inhibitor design.
NIH ExPorter
NIH ExPorter