Cryptosporidium parvum causes one of the opportunistic infections (0I) in AIDS patients for which no treatment is yet known. Although many drugs have been tested against this 0I. knowledge about the presence of target enzymes, biosynthetic pathways, and expression or regulation are virtually unknown. Our preliminary biochemical, molecular and ultrastructural data indicate that C. parvum has a mitochondrion for generating energy, and that an unusual fusion gene pyruvate: ferredoxin oxidoreductase/cytochrome P450 (PFO/P450) is important for its function. Unexpectedly. this apicomplexan PFO/P450 fusion most closely resembles that of Euglena gracilis. and in both, the C-terminus appears to function in methionine biosynthesis. C. parvum encodes S-adenosyl-methionine synthetase (SAMS) and S-adenosylhomocysteine hydrolase (SAHH). both of which are essential for its plant-like polyamine biosynthetic pathway. These genes appear to be functional since transcripts were detected by RT-PCR both in sporozoites and intracellular stages. Together this discovery suggests energy metabolism and polyamine biosynthesis in C. parvum are interrelated, thus different from that of humans. The long-term goal of this proposal is to determine whether these pathways in C. parvum can serve as a rational target for drug development.
The specific aims of this application are: 1. Characterize the C. parvum SAHH, SAMS & PFO/P450 gene structure, transcription and subcellular localization during the life cycle of the parasite. 2. Elucidate the function of SAHH, SAMS, & PFO in vitro using recombinant proteins. 3. Determine whether CpSAHH and CpPFO/P450 can serve as rational targets for drug development by screening specific analogs against C. parvum in vitro and in vivo. Completion of these specific aims will either support or refute our hypothesis that energy metabolism and polyamine biosynthesis in C. parvum are interrelated and sufficiently different from humans to be exploited for chemotherapy. If the pathway is verified as a parasite-specific target, then a major obstacle in the treatment of this OI in AIDS patients will have been overcome.
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| Slapeta, Jan; Stejskal, Frantisek; Keithly, Janet S (2003) Characterization of S-adenosylmethionine synthetase in Cryptosporidium parvum (Apicomplexa). FEMS Microbiol Lett 225:271-7 |
| LaGier, Michael J; Tachezy, Jan; Stejskal, Frantisek et al. (2003) Mitochondrial-type iron-sulfur cluster biosynthesis genes (IscS and IscU) in the apicomplexan Cryptosporidium parvum. Microbiology 149:3519-30 |
| Stejskal, Frantisek; Slapeta, Jan; Ctrnacta, Vlasta et al. (2003) A Narf-like gene from Cryptosporidium parvum resembles homologues observed in aerobic protists and higher eukaryotes. FEMS Microbiol Lett 229:91-6 |
