Regulators of Trypanosoma brucei Cell Cycle Progression and Differentiation Identified Using a Kinome-Wide RNAi Screen

Abstract

The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite's protein kinases (PKs) involved in the control of cellular proliferation and differentiation. To address this, a T. brucei kinome-wide RNAi cell line library was generated, allowing independent inducible knockdown of each of the parasite's 190 predicted protein kinases. Screening of this library using a cell viability assay identified ≥42 PKs that are required for normal bloodstream form proliferation in culture. A secondary screen identified 24 PKs whose RNAi-mediated depletion resulted in a variety of cell cycle defects including in G1/S, kinetoplast replication/segregation, mitosis and cytokinesis, 15 of which are novel cell cycle regulators. A further screen identified for the first time two PKs, named repressor of differentiation kinase (RDK1 and RDK2), depletion of which promoted bloodstream to procyclic form differentiation. RDK1 is a membrane-associated STE11-like PK, whilst RDK2 is a NEK PK that is essential for parasite proliferation. RDK1 acts in conjunction with the PTP1/PIP39 phosphatase cascade to block uncontrolled bloodstream to procyclic form differentiation, whilst RDK2 is a PK whose depletion efficiently induces differentiation in the absence of known triggers. Thus, the RNAi kinome library provides a valuable asset for functional analysis of cell signalling pathways in African trypanosomes as well as drug target identification and validation.

Author Summary

The African trypanosome, which is transmitted by the tsetse fly, causes the usually fatal disease Sleeping Sickness in humans and a wasting disease, called Nagana, in livestock in sub-Saharan Africa. There are no vaccines available against the diseases, and various problems are associated with current drug treatments (including toxicity to the patient and parasite drug resistance). Thus, it is important to identify essential parasite proteins that could be targeted by novel drugs. Protein kinases (PKs) are important cell signalling molecules, and are generally considered to have potential as drug targets. Here we report the construction of a library of trypanosome cell lines that allows us to specifically deplete each of the trypanosome's 190 PKs individually and analyse their function. Using this library, we show that ≥42 PKs are essential for proliferation of the mammalian-infective bloodstream form of the parasite (and thus have potential as drug targets), and demonstrate that 24 of these play important roles in coordinating cell division. We also shed light on how the parasite develops during its life cycle as it passes from the mammalian bloodstream form to the tsetse fly gut by identifying the first two PKs that regulate this life cycle developmental step.