Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation

Duncan, S. M., Myburgh, E. , Philipon, C., Brown, E., Meissner, M. , Brewer, J. and Mottram, J. (2016) Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation. Molecular Microbiology, 100(6), pp. 931-944. (doi:10.1111/mmi.13375) (PMID:26991545) (PMCID:PMC4913733)

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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 CRK3T178E 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 analysing essential genes in Leishmania.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Philipon, Dr Cintia and Duncan, Mr Samuel and Meissner, Professor Markus and Brewer, Professor James and Brown, Miss Elaine and Myburgh, Dr Elmarie and Mottram, Professor Jeremy
Authors: Duncan, S. M., Myburgh, E., Philipon, C., Brown, E., Meissner, M., Brewer, J., and Mottram, J.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Molecular Microbiology
Publisher:Wiley-Blackwell Publishing Ltd
ISSN:0950-382X
Published Online:13 April 2016
Copyright Holders:Copyright © 2016 The Authors
First Published:First published in Molecular Microbiology 100(6):931-944
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
611411Proteolysis and life cycle progression in LeishmaniaJeremy MottramMedical Research Council (MRC)MR/K019384/1III - PARASITOLOGY
665691In vivo screening resource to support drug development in parasitic diseaseJeremy MottramWellcome Trust (WELLCOME)104976/Z14/ZIII - PARASITOLOGY
371799The Wellcome Centre for Molecular Parasitology ( Core Support )Andrew WatersWellcome Trust (WELLCOME)104111/Z/14/Z &III - PARASITOLOGY