title: Genetic complementation in apicomplexan parasites.
abstract: A robust forward genetic model for Apicomplexa could greatly enhance functional analysis of genes in these important protozoan pathogens. We have developed and successfully tested a genetic complementation strategy based on genomic insertion in Toxoplasma gondii. Adapting recombination cloning to genomic DNA, we show that complementing sequences can be shuttled between parasite genome and bacterial plasmid, providing an efficient tool for the recovery and functional assessment of candidate genes. We show complementation, gene cloning, and biological verification with a mutant parasite lacking hypoxanthine-xanthine-guanine phosphoribosyltransferase and a T. gondii cDNA library. We also explored the utility of this approach to clone genes based on function from other apicomplexan parasites using Toxoplasma as a surrogate. A heterologous library containing Cryptosporidium parvum genomic DNA was generated, and we identified a C. parvum gene coding for inosine 5-monophosphate-dehydrogenase (IMPDH). Interestingly, phylogenetic analysis demonstrates a clear eubacterial origin of this gene and strongly suggests its lateral transfer from epsilon-proteobacteria. The prokaryotic origin of this enzyme might make it a promising target for therapeutics directed against Cryptosporidium.
authors.affiliation: Department of Cellular Biology and Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602, USA. striepen@cb.uga.edu
authors.forename: Boris
authors.initials: B
authors.lastname: Striepen
authors.forename: Michael W
authors.initials: MW
authors.lastname: White
authors.forename: Catherine
authors.initials: C
authors.lastname: Li
authors.forename: Michael N
authors.initials: MN
authors.lastname: Guerini
authors.forename: S-Banoo
authors.initials: SB
authors.lastname: Malik
authors.forename: John M
authors.initials: JM
authors.lastname: Logsdon
authors.forename: Chang
authors.initials: C
authors.lastname: Liu
authors.forename: Mitchell S
authors.initials: MS
authors.lastname: Abrahamsen
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D016785:Cryptosporidium parvum / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015723:Gene Library; D005816:Genetic Complementation Test; D007168:IMP Dehydrogenase / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D011995:Recombination, Genetic; D014122:Toxoplasma / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D004247:DNA; D007168:IMP Dehydrogenase