Date Published: June 25, 2018
Publisher: Public Library of Science
Author(s): Lindsay McDonald, Mathieu Cayla, Alasdair Ivens, Binny M. Mony, Paula MacGregor, Eleanor Silvester, Kirsty McWilliam, Keith R. Matthews, Kent L. Hill.
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.
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 . 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 [2–4]. The organisation of these signalling cascades is relatively well conserved in overall structure in eukaryotic organisms, although the individual receptors and transducer kinases and phosphatases are different. In particular, the ability of cells to become quiescent through exiting the cell cycle is a central feature of eukaryotic life, enabling cells to withstand periods of nutrient restriction, or to prepare for cell differentiation .
Genome-wide RNAi screens identify the genes whose silencing renders parasites resistant to an imposed selection. This approach was applied to identify genes that confer resistance to cell permeable cAMP, acting as an in vitro proxy for SIF-induced differentiation in vivo. The resulting screen identified approximately 30 genes, many of which were subsequently validated in individual RNAi lines, and tested for their inability to undergo natural stumpy formation in vivo. This confirmed the involvement of many of the hits from the original selection as molecules linked to developmental competence in bloodstream form trypanosomes. However, whilst the list of genes identified suggested the existence of a potential signalling pathway, the ordering and interactions between components of the pathway could not be assumed. Furthermore, it was unclear whether resistance to cell permeable cAMP was enacted through a simple processional linear pathway or whether there was more complexity. One established approach to address this question in conventional genetic systems has been to explore the ability of one molecular mutant to suppress a second mutant and thereby gain understanding of their relative positioning with respect to one another; this also often highlights direct molecular interactions between the respective molecules[36–38]. An alternative approach, epistasis or extragenic suppression, can use the ectopic expression of a wild type or mutant protein to restore a phenotype lost by mutation of another component in a pathway. This also orders the molecules with respect to one another, but does not necessarily imply direct molecular interaction. Here we have used extragenic suppression and phosphoproteomic analysis of a null mutant for a signalling component to dissect the pathway responsible for the generation of stumpy forms in response to the quorum sensing signal, stumpy induction factor, and assigned pairwise dependency relationships between several molecules involved in the process.