title: Characterization of a mitochondrion-like organelle in Cryptosporidium parvum.
abstract: Cryptosporidium parvum is a protozoan parasite that causes widespread diarrhoeal disease in humans and other animals and is responsible for large waterborne outbreaks of cryptosporidiosis. Unlike many organisms belonging to the phylum Apicomplexa, such as Plasmodium spp. and Toxoplasma gondii, there is no clinically proven drug treatment against this parasite. Aspects of the basic biology of C. parvum remain poorly understood, including a detailed knowledge of key metabolic pathways, its genome organization and organellar complement. Previous studies have proposed that C. parvum lacks a relic plastid organelle, or 'apicoplast', but that it may possess a mitochondrion. Here we characterize a mitochondrion-like organelle in C. parvum by (i) ultrastructural and morphological description (ii) localization of heterologous mitochondrial chaperonin antibody probes (iii) phylogenetic analysis of genes encoding mitochondrial transport proteins (iv) identification and analysis of mitochondrion-associated gene sequences. Our descriptive morphological analysis was performed by energy-filtering transmission electron microscopy (EFTEM) of C. hominis and C. parvum. The 'mitochondrion-like' organelle was characterized by labelling the structure with a heterologous mitochondrial chaperonin probe (hsp60) both in immunoelectron microscopy (IMEM) and immunofluorescence (IMF). Phylogenetic analysis of the mitochondrial import system and housekeeping components (hsp60 and hsp70-dnaK) suggested that the C. parvum mitochondrion-like organelle is likely to have descended from a common ancestral apicomplexan mitochondrion. We also identified a partial cDNA sequence coding for an alternative oxidase (AOX) gene, a component of the electron transport chain which can act as an alternative to the terminal mitochondrial respiratory complexes III and IV, which has not yet been reported in any other member of this phylum. Degenerate primers developed to identify selected mitochondrial genes failed to identify either cytochrome oxidase subunit I, or cytochrome b. Taken together, our data aim to provide new insights into the characterization of this Cryptosporidium organelle and a logical framework for future functional investigation.
authors.affiliation: Division of Infection and Immunity, Institute of Biomedical and Life Science, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: L
authors.initials: L
authors.lastname: Putignani
authors.forename: A
authors.initials: A
authors.lastname: Tait
authors.forename: H V
authors.initials: HV
authors.lastname: Smith
authors.forename: D
authors.initials: D
authors.lastname: Horner
authors.forename: J
authors.initials: J
authors.lastname: Tovar
authors.forename: L
authors.initials: L
authors.lastname: Tetley
authors.forename: J M
authors.initials: JM
authors.lastname: Wastling
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D018834:Chaperonin 60 / Q000378:metabolism; D003001:Cloning, Molecular; D016785:Cryptosporidium parvum / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009930:Organic Chemicals; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment
chemical_list: D002352:Carrier Proteins; D018834:Chaperonin 60; D016054:DNA, Protozoan; D005456:Fluorescent Dyes; D007211:Indoles; D009930:Organic Chemicals; C402795:SYTOX Green; C007293:DAPI