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

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          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          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.

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          Most cited references 51

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          The protein kinase complement of the human genome.

          We have catalogued the protein kinase complement of the human genome (the "kinome") using public and proprietary genomic, complementary DNA, and expressed sequence tag (EST) sequences. This provides a starting point for comprehensive analysis of protein phosphorylation in normal and disease states, as well as a detailed view of the current state of human genome analysis through a focus on one large gene family. We identify 518 putative protein kinase genes, of which 71 have not previously been reported or described as kinases, and we extend or correct the protein sequences of 56 more kinases. New genes include members of well-studied families as well as previously unidentified families, some of which are conserved in model organisms. Classification and comparison with model organism kinomes identified orthologous groups and highlighted expansions specific to human and other lineages. We also identified 106 protein kinase pseudogenes. Chromosomal mapping revealed several small clusters of kinase genes and revealed that 244 kinases map to disease loci or cancer amplicons.
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            Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers.

             H Hirumi,  K Hirumi (1989)
            Blood stream forms (BSF) of Trypanosoma brucei brucei GUT at 3.1 were propagated in vitro in the absence of feeder layer cells at 37 C, using a modified Iscove's medium (HMI-18). The medium was supplemented with 0.05 mM bathocuproine sulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM 2-mercaptoethanol, 1 mM sodium pyruvate. 0.16 mM thymidine, and 20% (v/v) Serum Plus (SP) (Hazleton Biologics, Lenexa, Kansas). The latter contained a low level of serum proteins (13 micrograms/ml). Each primary culture was initiated by placing 3.5-4 x 10(6) BSFs isolated from infected mice in a flask containing 5 ml of the medium (HMI-9) supplemented with 10% fetal bovine serum (FBS) and 10% SP. The cultures were maintained by replacing the medium every 24 hr for 5-7 days. During this period, many BSFs died. However, from day 4 onward, long slender BSFs increased in number. On days 5-7, trypanosome suspensions were pooled and cell debris was removed by means of diethylaminoethyl cellulose (DE52) column chromatography. Blood stream forms then were collected by centrifugation, resuspended in fresh medium at 7-9 x 10(5)/ml, and transferred to new flasks. Subcultures were maintained by readjusting the BSF density to 7-9 x 10(5)/ml every 24 hr. Concentrations of FBS were reduced gradually at 5-7-day intervals by alternating the amounts of FBS and SP in HMI-9 with 5% FBS and 15% SP, with 2% FBS and 18% SP, and finally with 20% SP (HMI-18). By this method, 2-3 x 10(6) VSFs/ml were obtained consistently every 24 hr. for more than 80 days.(ABSTRACT TRUNCATED AT 250 WORDS)
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              Construct design for efficient, effective and high-throughput gene silencing in plants.

              Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                January 2014
                January 2014
                16 January 2014
                : 10
                : 1
                Affiliations
                [1 ]Wellcome Trust Centre for Molecular Parasitology and Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
                [2 ]Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
                London School of Hygiene and Tropical Medicine, United Kingdom
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JCM TCH. Performed the experiments: NGJ EB EBT NJD. Analyzed the data: NGJ EBT NJD TCH JCM. Wrote the paper: NGJ TCH JCM.

                Article
                PPATHOGENS-D-13-02035
                10.1371/journal.ppat.1003886
                3894213
                24453978

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Counts
                Pages: 16
                Funding
                This work was supported by a Scottish Universities Life Sciences Alliance (SULSA) studentship to NGJ, an MRC/BBSRC studentship to EBT, and an MRC New Investigator Research Grant [GO900239] to TCH. The Wellcome Trust Centre for Molecular Parasitology is supported by core funding from the Wellcome Trust [085349]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Microbiology
                Parasitology
                Parasite Physiology

                Infectious disease & Microbiology

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