title: Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection.
abstract: The ROP1 gene of Toxoplasma gondii encodes a rhoptry protein that has been implicated in host cell invasion by this obligate intracellular protozoan. To further explore the function of this protein, we created a ROP1 deletion mutant by transfection with a plasmid encoding the bacterial chloramphenicol acetyltransferase (cat) gene flanked by ROP1 genomic sequences. Selection for chloramphenicol resistance yielded the desired ROP1-deleted or 'knock-out' mutant. Analysis of this mutant both in vitro and in vivo shows no significant alterations in growth rate, host specificity, invasiveness or virulence and thus the ROP1 gene is not obligatory for the RH strain, at least under the conditions tested. However, electron microscopy reveals that the mutant strain's rhoptries are altered in ultrastructure; they are thinner and homogeneously electron-dense compared with the thicker and normally mottled or honeycombed appearance of wild-type rhoptries. The knock-out mutant was rescued using co-transfection of a cosmid carrying the ROP1 gene together with a plasmid encoding a new selectable marker for T. gondii, the bleomycin resistance gene (ble) from Streptoalloteichus. Southern blot analysis showed that both DNAs were stably integrated into the Toxoplasma genome, although not into the ROPI locus. The resulting strain showed wild-type levels of ROP1 expression and rescue of the ultrastructural phenotype (i.e., the rhoptries returned to their normal, mottled appearance), thus establishing a cause/effect relationship between the absence of ROP1 and the electron-opacity. These results demonstrate the utility of the reverse genetic approach in the study of Toxoplasma gene function and provide a further selectable marker for such manipulations.
authors.affiliation: Department of Microbiology and Immonology, Stanford University School of Medicine, CA 94305-5402, USA.
authors.forename: D
authors.initials: D
authors.lastname: Soldati
authors.forename: K
authors.initials: K
authors.lastname: Kim
authors.forename: J
authors.initials: J
authors.lastname: Kampmeier
authors.forename: J F
authors.initials: JF
authors.lastname: Dubremetz
authors.forename: J C
authors.initials: JC
authors.lastname: Boothroyd
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002874:Chromosome Mapping; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D017353:Gene Deletion; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D005819:Genetic Markers; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D015388:Organelles / Q000648:ultrastructure; D010692:Phleomycins / Q000494:pharmacology*; D015800:Protozoan Proteins / Q000235:genetics* / Q000502:physiology; D014122:Toxoplasma / Q000187:drug effects* / Q000235:genetics* / Q000648:ultrastructure; D014774:Virulence / Q000235:genetics / Q000502:physiology
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D016054:DNA, Protozoan; D005819:Genetic Markers; D010692:Phleomycins; D015800:Protozoan Proteins; C065352:rhoptry associated protein, Plasmodium; D015500:Chloramphenicol O-Acetyltransferase