Abstract
Trypanosoma brucei, the agents of African trypanosomiasis, undergo density-dependent differentiation in the mammalian bloodstream to prepare for transmission by tsetse flies. This involves the generation of cell-cycle arrested, quiescent, stumpy forms from proliferative slender forms. The signalling pathway responsible for the quorum sensing response has been catalogued using a genome-wide selective screen, providing a compendium of signalling protein kinases phosphatases, RNA binding proteins and hypothetical proteins. However, the ordering of these components is unknown. To piece together these components to provide a description of how stumpy formation arises we have used an extragenic suppression approach. This exploited a combinatorial gene knockout and overexpression strategy to assess whether the loss of developmental competence in null mutants of pathway components could be compensated by ectopic expression of other components. We have created null mutants for three genes in the stumpy induction factor signalling pathway (RBP7, YAK, MEKK1) and evaluated complementation by expression of RBP7, NEK17, PP1-6, or inducible gene silencing of the proposed differentiation inhibitor TbTOR4. This indicated that the signalling pathway is non-linear. Phosphoproteomic analysis focused on one pathway component, a putative MEKK, identified molecules with altered expression and phosphorylation profiles in MEKK1 null mutants, including another component in the pathway, NEK17. Our data provide a first molecular dissection of multiple components in a signal transduction cascade in trypanosomes.
Highlights
Cells respond to their external environment in order to regulate their proliferation, developmental fate, specialisation or death
African trypanosome parasites respond to density sensing information in the bloodstream of their mammalian hosts to generate their transmission stage, the stumpy form
Each generated cell line was assessed in vivo for their ability to produce stumpy forms, this involving scoring the virulence of the parasites in mice, their accumulation as quiescent G1 arrested cells as parasite numbers increased, their morphological differentiation to stumpy forms and their expression of the stumpy form specific cell surface marker PAD1[31]
Summary
Cells respond to their external environment in order to regulate their proliferation, developmental fate, specialisation or death This can be in response to environmental cues such as temperature, pH or light, or can be driven by chemical signals generated by other cells of the same species or from competing or co-operating cells occupying the same niche [1]. To respond to such signals, single-celled and multicellular organisms have evolved elaborate signalling pathways in which surface receptors often transduce a signal to protein kinases and protein phosphatases, these transduction cascades eventually driving changes in gene expression either in their nucleus, or by generating phenotypic responses through changes in the abundance or activity of mRNAs or proteins [2,3,4]. The environmentally regulated control of virulence, transmission competence, tissue tropism or metabolic adaptation is poorly understood in eukaryotic microbial pathogens
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