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Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection
Published on: September 14, 2019
Crystallization of three key glycolytic enzymes of the opportunistic pathogen Cryptosporidium parvum
Olga Senkovich1, Haley Speed, Alexei Grigorian
1Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL-35294, USA.
Abstract:
Cryptosporidium parvum is one of the major causes of waterborne diseases worldwide. This protozoan parasite depends mainly on the anaerobic oxidation of glucose for energy production. In order to identify the differences in the three-dimensional structure of key glycolytic enzymes of C. parvum and its human host, we have expressed, purified and crystallized recombinant versions of three important glycolytic enzymes of the parasite, namely, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase. Lactate dehydrogenase has been crystallized in the absence and in the presence of its substrates and cofactors, while pyruvate kinase and glyceraldehyde 3-phosphate dehydrogenase were crystallized only in the apo-form. X-ray diffraction data have been collected for all crystals.
Insights
Researchers studied key energy-producing enzymes in Cryptosporidium parvum, a cause of waterborne illness. Structural analysis of these enzymes may reveal differences between the parasite and humans, aiding drug development.
Area of Science:
- Biochemistry
- Parasitology
- Structural Biology
Background:
- Cryptosporidium parvum is a significant cause of global waterborne diseases.
- This protozoan parasite relies on anaerobic glucose metabolism for energy.
- Understanding structural differences in parasitic enzymes compared to human hosts is crucial for therapeutic strategies.
Purpose of the Study:
- To investigate the three-dimensional structures of key glycolytic enzymes in Cryptosporidium parvum.
- To identify structural distinctions between C. parvum enzymes and their human host counterparts.
- To provide a structural basis for developing targeted anti-parasitic agents.
Main Methods:
- Expression and purification of recombinant glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase from C. parvum.
- Crystallization of these enzymes in apo-form and, for lactate dehydrogenase, in the presence of substrates and cofactors.
- Collection of X-ray diffraction data for all crystallized enzyme forms.
Main Results:
- Recombinant versions of three key glycolytic enzymes were successfully produced and crystallized.
- Lactate dehydrogenase was crystallized in multiple states, offering insights into its conformational flexibility.
- X-ray diffraction data were obtained, paving the way for detailed structural determination.
Conclusions:
- The structural characterization of C. parvum glycolytic enzymes has been initiated.
- These structural insights are vital for understanding parasite metabolism and host-pathogen interactions.
- The identified structural differences can inform the design of novel therapeutics against cryptosporidiosis.
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