Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection

Dominique Soldati, Kami Kim, Jennifer Kampmeier, Jean Francois Dubremetz, John C. Boothroyd

Research output: Contribution to journalArticle

73 Citations (Scopus)

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 ROP1 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.

Original languageEnglish (US)
Pages (from-to)87-97
Number of pages11
JournalMolecular and Biochemical Parasitology
Volume74
Issue number1
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

Phleomycins
Toxoplasma
Genes
Transfection
Plasmids
Chloramphenicol Resistance
Electrons
Reverse Genetics
Cosmids
Chloramphenicol O-Acetyltransferase
Host Specificity
Bleomycin
Southern Blotting
Virulence
Electron Microscopy
Proteins
Genome
Phenotype
DNA
Growth

Keywords

  • Phleomycin resistance gene
  • Rhoptry protein
  • Stable transformation
  • Toxoplasma gondii

ASJC Scopus subject areas

  • Molecular Biology
  • Parasitology

Cite this

Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection. / Soldati, Dominique; Kim, Kami; Kampmeier, Jennifer; Dubremetz, Jean Francois; Boothroyd, John C.

In: Molecular and Biochemical Parasitology, Vol. 74, No. 1, 1995, p. 87-97.

Research output: Contribution to journalArticle

Soldati, Dominique ; Kim, Kami ; Kampmeier, Jennifer ; Dubremetz, Jean Francois ; Boothroyd, John C. / Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection. In: Molecular and Biochemical Parasitology. 1995 ; Vol. 74, No. 1. pp. 87-97.
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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 ROP1 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.",
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