Deletion of a novel protein kinase with PX and FYVE‐related domains increases the rate of differentiation of Trypanosoma brucei

Abstract

SummaryGrowth control of African trypanosomes in the mammalian host is coupled to differentiation of a non-dividing life cycle stage, the stumpy bloodstream form. We show that a protein kinase with novel domain architecture is important for growth regulation. Zinc finger kinase (ZFK) has a kinase domain related to RAC and S6 kinases flanked by a FYVErelated zinc finger and a phox (PX) homology domain. To investigate the function of the kinase during cyclical development, a stable transformation procedure for bloodstream forms of differentiationcompetent (pleomorphic) Trypanosoma brucei strains was established. Deletion of both allelic copies of ZFK by homologous recombination resulted in reduced growth of bloodstream-form parasites in culture, which was correlated with an increased rate of differentiation to the non-dividing stumpy form. Growth and differentiation rates were returned to wild-type level by ectopic ZFK expression. The phenotype is stage-specific, as growth of procyclic (insect form) trypanosomes was unaffected, and Dzfk/Dzfk clones were able to undergo full cyclical development in the tsetse fly vector. Deletion of ZFK in a differentiation-defective (monomorphic) strain of T. brucei did not change its growth rate in the bloodstream stage. This suggests a function of ZFK associated with the trypanosomes' decision between either cell cycle progression, as slender bloodstream form, or differentiation to the non-dividing stumpy form.

The Trypanosoma brucei Cyclin, CYC2, Is Required for Cell Cycle Progression through G1 Phase and for Maintenance of Procyclic Form Cell Morphology

“…Transfection of T. brucei-Culture and transfection of T. brucei were carried out essentially as described previously (18). Where necessary, hygromycin was added at a concentration of 50 g ml Ϫ1 (procyclic cells) or 5 g ml Ϫ1 (bloodstream form cells), G418 at 10 g ml Ϫ1 (procyclic cells) or 2.5 g ml Ϫ1 (bloodstream form cells), zeocin at 10 g ml Ϫ1 (procyclic cells), and phleomycin at 2.5 g ml Ϫ1 (bloodstream form cells).…”

Kinetoplastid guide RNA biogenesis is dependent on subunits of the mitochondrial RNA binding complex 1 and mitochondrial RNA polymerase

“…About 3 3 10 7 cells of exponentially growing culture were used for transfection, following the protocol of Vassella et al (2001) with minor modifications. Transfectants were selected with 1.25 mg/mL phleomycin and cloned by limiting dilution.…”

Cold shock and regulation of surface protein trafficking convey sensitization to inducers of stage differentiation in Trypanosoma brucei

“…For T7-polymerase-driven protein expression the transgenic T. brucei clone 13-90 (Wirtz et al 1999) was used. Culture conditions and transfection protocols are detailed in Vassella et al (2001b) and McCulloch et al (2004). Bloodstreamstage trypanosomes were grown in HMI-9 medium (Hirumi and Hirumi 1989).…”