Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation. Issue 6 (13th April 2016)

• Record Type: Journal Article
• Authors: Duncan, Samuel M.; Myburgh, Elmarie; Philipon, Cintia; Brown, Elaine; Meissner, Markus; Brewer, James; Mottram, Jeremy C.
• Is Part Of: Molecular microbiology. Volume 100:Issue 6(2016)
• Page Start: 931
• Page End: 944
• Publication Date: 2016-04-13
• Subjects: Molecular microbiology -- Periodicals; 572.829
• DOI: 10.1111/mmi.13375
• Languages: English
• ISSNs: 0950-382X

Abstract

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 . Abstract : Many protein kinases are essential post‐transcriptional regulators of cell cycle and life cycle progression in Leishmania . To investigate their function, we have developed a conditional method for deletion of essential genes, based on diCre‐lox mediated excision, which we have applied to the cdc2‐related protein kinase CRK3 . We demonstrate that CRK3 activity is essential for cell cycle progression through mitosis, and show that conditional deletion of the gene in mammalian infectious L. mexicana promastigotes prevents proliferation in a murine model of infection.