Non-linear hierarchy of the quorum sensing signalling pathway in bloodstream form African trypanosomes

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.

An atypical DYRK kinase connects quorum-sensing with posttranscriptional gene regulation inTrypanosoma brucei

“…We initially confirmed the role for TbDYRK in differentiation competence described by [9] and [8]. A wild-type differentiation competent cell line and a previously validated TbDYRK knock-out (KO) cell line [8] were assayed in vitro using the cell permeable cyclic AMP analogue, 8-pCPT-cAMP, that promotes the expression of some characteristics of stumpy forms ([21, 22]). As expected, the wild-type cells differentiated in response to treatment, causing a 60% growth inhibition at 96 hours compared to untreated cells, whereas the TbDYRK KO cells had reduced differentiation competence, with only 23% growth inhibition at the same timepoint (Figure 2a).…”

“…In contrast, of the seven DYRKs identified in the T. brucei kinome [3], four present unconventional activation motifs, of which two include a histidine (Tb927.10.15020 and Tb927.5.1650), suggesting a particular mode of regulation in trypanosomes more developed than in other species. Given the association with the DYRK family, we henceforth refer to the protein encoded by Tb927.10.15020 as TbDYRK (previously termed ‘YAK’ in [9] and [8]).…”

“…This differentiation is triggered by a quorum-sensing (QS)-like mechanism where parasites respond to the accumulation of a stumpy-induction factor [5–7]. Once the signal is received, it is transduced via a non-linear hierarchical signalling pathway [8] comprising at least 30 molecules [9]. This includes signal processing molecules, protein kinases and phosphatases and post-transcriptional gene expression regulators as well as additional proteins of unknown function.…”

“…One of the components involved in the differentiation process is a molecule related to the protein kinase Yak sub family [8, 9]. Yak kinases belong to the dual-specificity yak-related kinases (DYRK) family included in the CMGC group, which is over represented in trypanosomatids compared to humans [10].…”

Identification of clinically approved small molecules that inhibit growth and promote surface remodeling in the African trypanosome

“…Much progress has been made in elucidating the quorum sensing signaling pathways that are necessary to trigger parasite differentiation from the bloodstream form to the insect form via the stumpy intermediate (10)(11)(12). It is also clear that RNA Binding Proteins (RBPs) play an important role in maintaining life cycle stage-specific proteins and triggering differentiation events (13)(14)(15)(16).…”