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Molecular characterization of ribonucleotide reductase from Cryptosporidium parvum
Donna E Akiyoshi1, Ramaswamy Balakrishnan, Christian Huettinger
1Division of Infectious Diseases, Tufts University School of Veterinary Medicine, North Grafton, MA 01536, USA. donna.akiyoshi@tufts.edu
Abstract:
The Apicomplexan enteric parasite, Cryptosporidium, infects a broad range of mammals, birds, fish and reptiles. Cryptosporidium parvum, the principal causative agent of human cryptosporidiosis, has emerged as a major contributor to waterborne outbreaks of this disease. The absence of effective drugs against C. parvum has necessitated the search for new drug targets. One attractive class of target enzymes is ribonucleotide reductase, an essential enzyme required for the de novo synthesis of deoxyribonucleotides. We report the cloning and sequencing of the small and large subunits of ribonucleotide reductase from a C. parvum genotype 2 isolate, GCH1. Southern blot analysis showed that these subunits are encoded by single copy genes. Both subunits have been expressed as recombinant proteins in Escherichia coli.
Insights
Cryptosporidium parvum causes human cryptosporidiosis, driving a need for new drugs. Researchers cloned and expressed key enzyme subunits, identifying potential drug targets for this parasite.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Apicomplexan enteric parasite Cryptosporidium infects various hosts, with Cryptosporidium parvum causing human cryptosporidiosis and waterborne disease outbreaks.
- The lack of effective treatments for C. parvum infections highlights the urgent need for novel therapeutic strategies and drug targets.
- Ribonucleotide reductase, essential for deoxyribonucleotide synthesis, is a promising target for antiparasitic drug development.
Purpose of the Study:
- To clone and sequence the small and large subunits of ribonucleotide reductase from Cryptosporidium parvum.
- To investigate the gene structure of ribonucleotide reductase subunits in C. parvum.
- To express recombinant ribonucleotide reductase subunits for further study.
Main Methods:
- Cloning and sequencing of ribonucleotide reductase subunits from C. parvum genotype 2 (GCH1).
- Southern blot analysis to determine gene copy number.
- Expression of recombinant proteins in Escherichia coli.
Main Results:
- The small and large subunits of ribonucleotide reductase from C. parvum were successfully cloned and sequenced.
- Southern blot analysis confirmed that the genes encoding these subunits are single copy.
- Both recombinant subunits were expressed in E. coli.
Conclusions:
- The cloning, sequencing, and expression of C. parvum ribonucleotide reductase subunits provide a foundation for developing new drugs targeting this essential enzyme.
- Identifying and characterizing essential enzymes like ribonucleotide reductase is crucial for discovering novel therapeutic interventions against cryptosporidiosis.