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

Samuel M Duncan,James Brewer,Markus Meissner,Elmarie Myburgh,Cintia Philipon,Elaine Brown,Jeremy C Mottram

doi:10.1111/mmi.13375

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.

Highlights

The leishmaniases, diseases caused by protozoan parasites of the genus Leishmania, have diverse clinical manifestations dependent on the species and host immune response
Leishmania mexicana has a large family of cyclindependent 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

Summary

Introduction

The leishmaniases, diseases caused by protozoan parasites of the genus Leishmania, have diverse clinical manifestations dependent on the species and host immune response. Protein kinases elicit pronounced effects on the Leishmania cell cycle by regulation of cell signalling pathways, and a number of protein kinases have been identified that are essential for promastigote viability (Dacher et al, 2014; Wang et al, 2005). The use of CDK inhibitors in cancer therapy (Cicenas and Valius, 2011; Knapp and Sundstro€m, 2014) and the relative expansion of this protein family in Leishmania relative to other unicellular organisms distinguishes them as suitable drug targets. The CDKrelated kinase CRK3 has been demonstrated as being important for regulation of the L. mexicana promastigote cell cycle by existing genetic manipulation techniques and cell cycle arrest following treatment with CDK inhibitors (Hassan et al, 2001; Grant et al, 1998; 2004).

Results

Discussion

Conclusion