Ordering components of the slender to stumpy signalling pathway in Trypanosoma brucei

Abstract

In the mammalian bloodstream, the protozoan parasite Trypanosoma brucei undergoes differentiation from proliferative slender forms to arrested, transmissible, stumpy forms. This transition is associated with extensive cytological and metabolic changes that promote survival in the tsetse midgut, and also influences infection dynamics within the mammalian host. A number of genes involved in this transformation were recently identified using an RNAi library screen for resistance to pCPTcAMP, a membrane-permeable cyclic AMP analogue that induces differentiation. These molecules, referred to here as posST (positive mediators of STumpy formation), were thereafter validated to regulate the slender to stumpy transition, with many of them apparently comprising part of a signal transduction and effector pathway. However, it is unknown how these proteins act in relation to one another or are ordered within the pathway. To this end, null mutants were created for several posST components in differentiation-competent pleomorphic trypanosomes and, in this genetic background, other members of the predicted pathway expressed to test their ability to restore stumpy formation. Analysis of distinct combinations has been used to build a preliminary pathway structure model for the signalling events underlying trypanosome quorum sensing. In addition, phosphoproteomic analysis of two null mutants has revealed downstream signalling effects of two posST kinases, MEKK1 and YAK. A similar extragenic suppression approach was also applied to explore the interaction between the identified drivers of stumpy formation and the target of rapamycin kinase, TOR4, which has previously been shown to act as a negative regulator of stumpy formation in monomorphs. Dual ablation of TOR4 and posST components revealed insight into the intersection of stumpy-promoting and stumpy-inhibiting pathways. Finally, a chemical-genetic approach was used to investigate the posST pathway using two differentiation-inducing compounds: the previously studied E667, and GKI7, newly identified from a kinase inhibitor set. RNAi lines for different posST components were tested for their ability to undergo development in the presence of these compounds. An RNAi library screen using GKI7 identified putative new mediators of stumpy formation.