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Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in L eishmania mexicana cell cycle regulation
Author(s) -
Duncan Samuel M.,
Myburgh Elmarie,
Philipon Cintia,
Brown Elaine,
Meissner Markus,
Brewer James,
Mottram Jeremy C.
Publication year - 2016
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.13375
Subject(s) - biology , cyclin dependent kinase 1 , cell cycle , cyclin dependent kinase , complementation , microbiology and biotechnology , cyclin , gene , kinase , mutant , transgene , genetics
Summary 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 .