title: Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation.
abstract: Leishmania mexicana has a large family of cyclin-dependent kinases (CDKs) that reflect the complex interplay between cell cycle and life cycle progression. Evidence from previous studies indicated that Cdc2-related kinase 3 (CRK3) in complex with the cyclin CYC6 is a functional homologue of the major cell cycle regulator CDK1, yet definitive genetic evidence for an essential role in parasite proliferation is lacking. To address this, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) to target CRK3 and elucidate its role in the cell cycle of L. mexicana. Induction of diCre activity in promastigotes with rapamycin resulted in efficient deletion of floxed CRK3, resulting in G2/M growth arrest. Co-expression of a CRK3 transgene during rapamycin-induced deletion of CRK3 resulted in complementation of growth, whereas expression of an active site CRK3(T178E) mutant did not, showing that protein kinase activity is crucial for CRK3 function. Inducible deletion of CRK3 in stationary phase promastigotes resulted in attenuated growth in mice, thereby confirming CRK3 as a useful therapeutic target and diCre as a valuable new tool for analyzing essential genes in Leishmania.
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.
authors.forename: Samuel M
authors.initials: SM
authors.lastname: Duncan
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.|Centre for Immunology and Infection, Department of Biology, University of York, Wentworth Way, Heslington, York, YO10 5DD, UK.
authors.forename: Elmarie
authors.initials: E
authors.lastname: Myburgh
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.
authors.forename: Cintia
authors.initials: C
authors.lastname: Philipon
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.|Centre for Immunology and Infection, Department of Biology, University of York, Wentworth Way, Heslington, York, YO10 5DD, UK.
authors.forename: Elaine
authors.initials: E
authors.lastname: Brown
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.
authors.forename: Markus
authors.initials: M
authors.lastname: Meissner
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.
authors.forename: James
authors.initials: J
authors.lastname: Brewer
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.|Centre for Immunology and Infection, Department of Biology, University of York, Wentworth Way, Heslington, York, YO10 5DD, UK.
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Mottram
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics; D059447:Cell Cycle Checkpoints / Q000235:genetics; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D059565:G2 Phase Cell Cycle Checkpoints / Q000235:genetics; D017353:Gene Deletion; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D007894:Leishmania mexicana / Q000166:cytology* / Q000201:enzymology / Q000235:genetics*; D016773:Leishmaniasis, Cutaneous / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D050737:Proto-Oncogene Proteins c-crk / Q000235:genetics* / Q000378:metabolism*; D059386:Reverse Genetics / Q000379:methods; D020123:Sirolimus / Q000494:pharmacology
chemical_list: D016213:Cyclins; D050737:Proto-Oncogene Proteins c-crk; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases; C045073:Cre recombinase; D019426:Integrases; D020123:Sirolimus