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.

Protein diversity in discrete structures at the distal tip of the trypanosome flagellum

“…Tb927.11.8150 is a member of the ubiquitous Unc-51-like kinase 4 (ULK4) family, and we refer to it as FCP5/TbULK4. The T. brucei genome contains a second gene coding for an ULK protein, Tb927.11.4470, a member of the Fused family (24). This FCP6/TbFused protein tagged with YFP was also found to localize specifically to the new flagellum tip of dividing cells (SI Appendix, Fig.…”

Cancer in the parasitic protozoans Trypanosoma brucei and Toxoplasma gondii

“…For example, the protozoan parasite must have genes that function to control the cell cycle to ensure correct coordination of multiplication. In the case of T. brucei, the molecular components of cell cycle control are well characterized (31). At least 45 genes, mostly encoding protein kinases, have been identified that are essential for parasite growth and replication.…”

“…However, it must be recognized that a coevolutionary journey between host and parasite should ensure no death of the host, upon which it depends, before finding their way to a new host. Despite the dependence of a protozoan parasite on the host, the innate cellular mechanisms, such as control of the cell cycle and differentiation, are analogous in protozoan, metazoan, or human cells (31). Failure of these processes (uncontrolled parasite growth and incorrect differentiation) could lead to further adverse pathogenesis to the host caused by an increased parasite load, causing cell and tissue destruction-a similar outcome to that generated by invasive cancer cells.…”

Nuclear DNA replication and repair in parasites of the genusLeishmania: Exploiting differences to develop innovative therapeutic approaches

“…DNA replication initiator factor. Associates with GINS and MCM complexes, and travels with the replication fork DDK (CDC7/DBF4) CDC7 CDC7 Not found (Jones et al, 2014;Parsons et al, 2005;Tiengwe et al, 2012a) Catalytic subunit of DDK (CDC7/DBF4), required for origin firing and replication fork progression in mitotic S phase DBF4 DBF4 Not found (Tiengwe et al, 2012a) Regulatory subunit of DDK (CDC7/DBF4), required for origin firing and replication fork progression in mitotic S phase TICRR (Treslin) SLD3 Not found (Dang & Li, 2011;Genois et al, 2014;Li, 2012) Genes nomenclature has been retrieved from HUGO gene nomenclature committee (http://www.genenames.org/) for H. sapiens; SGD: Saccharomyces genome database (http://www.yeastgenome.org/) for S. cerevisiae; and GeneDB (http://www.genedb.org). For Leishmania, genes names designation follows the guidelines for Trypanosoma and Leishmania genetic nomenclature (Clayton et al, 1998).…”

“…This could be explained by extensive sequence divergence. Indeed, several kinases involved in replication have been described in trypanosomatids (Jones et al, 2014;Parsons et al, 2005). It is possible that one or more of these kinases is the functional homolog of CDC7.…”