title: Gene targeting demonstrates that the Plasmodium berghei subtilisin PbSUB2 is essential for red cell invasion and reveals spontaneous genetic recombination events.
abstract: The Plasmodium merozoite proteases involved in the crucial process of erythrocyte invasion are promising targets for novel malaria control strategies. We report here the characterization of the subtilisin-like protease SUB2 from the rodent parasites Plasmodium berghei and Plasmodium yoelii, leading the way to in vivo functional studies of this enzyme. The kinetics of expression and subcellular localization imply a central role for SUB2 in erythrocyte invasion. Through the use of gene targeting strategies, we assessed the relevance of P. berghei SUB2 for the intraerythrocytic cycle. The selection of recombinant Pbsub2-TrimycDuoXpress-tagged parasites and the proper expression of the modified coding region demonstrate that the Pbsub2 locus is accessible to genetic modifications. However, Pbsub2 knock-out parasites were not recovered, confirming the importance of PbSUB2 for P. berghei merozoite stages, and supporting the fact that its Plasmodium falciparum SUB2 orthologue is an attractive drug target candidate. Finally, we identify revertant parasites that have lost the integrated selection cassette while conserving a Pbsub2-tagged gene. These spontaneous reversion events should overcome the scarcity of selectable markers available for this parasite, giving access to multiple gene tagging strategies, which, together with the validation of a TrimycDuoXpress tag, would represent valuable new tools for studying the biology of P. berghei.
authors.affiliation: Unité de Biologie des Interactions Hôte-Parasite, Institut Pasteur/CNRS URA 2581, 25 Rue du Dr Roux, 75015 Paris, France.
authors.forename: Pierrick
authors.initials: P
authors.lastname: Uzureau
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Barale
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Janse
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Waters
authors.forename: Catherine Braun
authors.initials: CB
authors.lastname: Breton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D004912:Erythrocytes / Q000469:parasitology*; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods; D017125:Genes, Protozoan; D051379:Mice; D008969:Molecular Sequence Data; D010962:Plasmodium berghei / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D014162:Transfection
chemical_list: D016054:DNA, Protozoan; D011994:Recombinant Proteins; D013381:Subtilisins