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High throughput screening for chemical inducers of stumpy formation in Trypanosoma brucei

ABSTRACT: During the bloodstream stage of the Trypanosoma brucei lifecycle, the parasite exists as the proliferative slender-form or the non-proliferative, transmissible, stumpy-form. The transition from the slender to stumpy-form is stimulated by a density-dependent mechanism and is important in infection dynamics, ordered antigenic variation and disease transmissibility. Here, we use a monomorphic reporter cell line in a whole-cell fluorescence-based assay to screen over 6000 small molecules from a kinase-focussed compound library for their ability to induce stumpy-like formation in a high-throughput screening programme. This identified one compound able to induce modest, yet specific, changes in gene expression indicative of a partial differentiation to stumpy forms. This not only provides a potential tool for the further understanding of stumpy formation, but also demonstrates the use of high throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes.

ORGANISM(S): Trypanosoma brucei brucei

PROVIDER: GSE46483 | GEO | 2014/01/21

SECONDARY ACCESSION(S): PRJNA200624

REPOSITORIES: GEO

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Project description:The protozoan parasites Trypanosoma brucei spp. are responsible for important human and livestock diseases in sub Saharan Africa. In the mammalian blood, two developmental forms of the parasite exist: proliferative ?slender? forms and transmissible ?stumpy? forms that are quiescent, awaiting uptake in a tsetse fly bloodmeal. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission. The response is triggered by an elusive ?stumpy induction factor? (SIF) whose intracellular signaling pathway is also completely uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains cannot respond to SIF, but can generate forms with stumpy characteristics when exposed to cell permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNAi library screen to identify the signaling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to cell permeable cAMP or AMP analogues to select cells that remained proliferative and so were unresponsive to these signals. Genome-wide ion torrent-based RNA interference Target sequencing (RIT-seq) identified a cohort of genes implicated in all steps of the signaling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. The identified genes at each step have been validated in cells naturally capable of stumpy formation, confirming their role in SIF-induced density sensing and cellular quiescence. RNA was isolated from AnTat1.1 cells grown in mice with or without doxycycline. RBP7 (Tb927.10.12100) RNAi lines were grown in mice with or without doxycycline. Since the RBP7 RNAi is leaky (six independent cell lines were analysed and all were significantly leaky), transcripts that showed elevated or reduced abundance in both RNAi induced and uninduced populations were identified in comparison to the AnTat1.1 control. A similar approach was adopted for the RBP7 over-expressing lines.

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High throughput screening for chemical inducers of stumpy formation in Trypanosoma brucei

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