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Surprisingly, unlike most Apicomplexa, Cryptosporidium parvum appears to lack a plastid genome. Primers based upon the highly conserved plastid smallor large-subunit rRNA (SSU/LSU rRNA) and the tufA-tRNAPhe genes of other members of the phylum Apicomplexa failed to amplify products from intracellular stages of C. parvum, whereas products were obtained from the plastid-containing apicomplexans Eimeria bovis and Toxoplasma gondii, as well as the plants Allium stellatum and Spinacia oleracea. Dot-blot hybridization of sporozoite genomic DNA (gDNA) supported these PCR results. A T. gondii plastid-specific set of probes containing SSU/LSU rRNA and tufAtRNA Phe genes strongly hybridized to gDNA from a diverse group of plastidcontaining organisms including three Apicomplexa, two plants, and Euglena gracilis, but not to those without this organelle including C. parvum, three kinetoplastids, the yeast Saccharomyces cerevisiae, mammals and the eubacterium Escherichia coli. Since the origin of the plastid in other apicomplexans is postulated to be the result of a secondary symbiogenesis of either a red or a green alga, the most parsimonious explanation for its absence in C. parvum is that it has been secondarily lost. If confirmed, this would indicate an alternative evolutionary fate for this organelle in one member of the Apicomplexa. It also suggests that unlike the situation with other diseases caused by members of the Apicomplexa, drug development against cryptosporidiosis targeting a plastid genome or metabolic pathways associated with it may not be useful.

Most eukaryotes perform the oxidative decarboxylation of pyruvate in mitochondria using pyruvate dehydrogenase (PDH). Eukaryotes that lack mitochondria also lack PDH, using instead the O(2)-sensitive enzyme pyruvate : ferredoxin oxidoreductase (PFO), which is localized either in the cytosol or in hydrogenosomes. The facultatively anaerobic mitochondria of the photosynthetic protist Euglena gracilis constitute a hitherto unique exception in that these mitochondria oxidize pyruvate with the O(2)-sensitive enzyme pyruvate : NADP oxidoreductase (PNO). Cloning and analysis of Euglena PNO revealed that the cDNA encodes a mitochondrial transit peptide followed by an N-terminal PFO domain that is fused to a C-terminal NADPH-cytochrome P450 reductase (CPR) domain. Two independent 5.8-kb full-size cDNAs for Euglena mitochondrial PNO were isolated; the gene was expressed in cultures supplied with 2% CO(2) in air and with 2% CO(2) in N(2). The apicomplexan Cryptosporidium parvum was also shown to encode and express the same PFO-CPR fusion, except that, unlike E. gracilis, no mitochondrial transit peptide for C. parvum PNO was found. Recombination-derived remnants of PNO are conserved in the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe as proteins involved in sulfite reduction. Notably, Trypanosoma brucei was found to encode homologs of both PFO and all four PDH subunits. Gene organization and phylogeny revealed that eukaryotic nuclear genes for mitochondrial, hydrogenosomal, and cytosolic PFO trace to a single eubacterial acquisition. These findings suggest a common ancestry of PFO in amitochondriate protists with Euglena mitochondrial PNO and Cryptosporidium PNO. They are also consistent with the view that eukaryotic PFO domains are biochemical relics inherited from a facultatively anaerobic, eubacterial ancestor of mitochondria and hydrogenosomes.

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