This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: The goal of our new NIH project is to elucidate the structure and function of the mitochondrion (mit) of AIDS-associated Cryptosporidium parvum. Our data indicate that C. parvum possesses an acristate, ribosome-studded mit and crystalloid body (CB) posterior to the nucleus that has cardiolipin and oligomycin- and cyanide-sensitive membrane potentials (DY). The nucleus of C. parvum also encodes mit-specific adenylate kinase 2, cpn60, valyl t-RNA synthase, and the iron sulfur cluster (Isc) genes IscS, IscU, and ferredoxin (Fdx), whose iron sulfur proteins are asembled and mature within eukaryotic mit. One of these, IscS has a typical mit signal peptide that has been used to target GFP to yeast mitochondria. C. parvum also contains a biochemical relic that appears to link pyruvate metabolism in mit-bearing protists with amitochondriate protists. Most aerobic protists oxidatively decarboxylate pyruvate within a mit using pyruvate dehydrogenase, whereas most anaerobic protists use pyruvate ferredoxin oxidoreductase (PFO). Euglena gracilis is exceptional using instead a pyruvate:NADP+ oxidoreductase (PNO) which is fused to a NADPH:cytochrome P-450 reductase (CPR) within its facultatively anaerobic mit. Interestingly, C. parvum also encodes and expresses this PNO:CPR fused protein, although its subcellular location is as yet unknown. We plan to test the hypothesis that C. parvum has a functional, and structurally relic mit that differs substantially from that of most eukaryotes, including humans, which may serve as a new drug target. The RVBC is needed to resolve the structural relationships between the relic mit, nuclear membranes, and CB immediately adjacent to the mit. Through traditional TEM, we have been able to make some assumptions about the structure and interrelationship of these organelles, but a more detailed image analysis is essential if we want to show definitively whether the mit and crystalloid body are both involved in energy metabolism of this parasite. Specifically, we would like to determine the nature of the membranous whorls in the relic mit, the apparent flattened cristae in the CB, and most specifically to determine if these two organelles are both part of an unusual mit. Furthermore, it already appears that the ribosomes surrounding the organelle also surround the nuclear membrane. We need to understand the juxtaposition of the ER and NM both in situ and in isolated organelles. In some respects, the CB of C. parvum resembles the unusual paracrystalline organization with cubic symmetry of the mit from Chaos carolinensis, and for that reason we think it might also perform mit functions. The sizes of these organelles suggests that a combination of imaging techniques available in the RVBC would be extremely helpful in resolving the structural complexity of the Cp mit, CB, and surrounding membranes. The following paper was published: + Keithly JS, Langreth SG, Buttle KF, Mannella CA. (2005) Electron tomographic and ultrastructural analysis of the Cryptosporidium parvum relict mitochondrion, its associated membranes, and organelles. J. Eukaryotic Microbiology, 52, 132-140. Additional visualization work was done on an existing reconstruction of a paracrystalline body, in order to investigate segmentation by means of new filtering and isosurface techniques. Figures from existing data were prepared for use in invited book chapters. Animations of surface-rendered models were made for presentation at future meetings. Chyongere Hsieh made four cryo-tomographic reconstructions of frozen-hydrated whole mounts of C. parvum sporozoites.
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