The protozoan parasites Trypanosoma brucei spp. cause important human and livestock diseases in sub Saharan Africa. In the mammalian blood, two developmental forms of the parasite exist: proliferative ‘slender’ forms and arrested ‘stumpy’ forms that are responsible for transmission to tsetse flies. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing (QS) in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission1. This response is triggered by an elusive ‘stumpy induction factor’ (SIF) whose intracellular signaling pathway is also uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains respond inefficiently 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 8-(4-chlorophenylthio)-cAMP (pCPTcAMP) or 8-pCPT-2′-O-Me-5′-AMP to select cells that were unresponsive to these signals and hence remained proliferative. Genome-wide ion torrent-based RNA interference Target sequencing identified cohorts of genes implicated in each step of the signaling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. Genes at each step were independently validated in cells naturally capable of stumpy formation, confirming their role in density sensing in vivo, whilst the putative RNA-binding protein, RBP7, was required for normal QS and promoted cell-cycle arrest and transmission competence when overexpressed. This study reveals that QS signaling in trypanosomes shares similarities to fundamental quiescence pathways in eukaryotic cells, its components providing targets for QS-interference based therapeutics.

African trypanosomes are sustained in the bloodstream of their mammalian hosts by their extreme capacity for antigenic variation. However, for life cycle progression, trypanosomes also must generate transmission stages called stumpy forms that are pre-adapted to survive when taken up during the bloodmeal of the disease vector, tsetse flies. These stumpy forms are rather different to the proliferative slender forms that maintain the bloodstream parasitaemia. Firstly, they are non proliferative and morphologically distinct, secondly, they show particular sensitivity to environmental cues that signal entry to the tsetse fly and, thirdly, they are relatively robust such that they survive the changes in temperature, pH and proteolytic environment encountered within the tsetse midgut. These characteristics require regulated changes in gene expression to pre-adapt the parasite and the use of environmental sensing mechanisms, both of which allow the rapid initiation of differentiation to tsetse midgut procyclic forms upon transmission. Interestingly, the generation of stumpy forms is also regulated and periodic in the mammalian blood, this being governed by a density-sensing mechanism whereby a parasite-derived signal drives cell cycle arrest and cellular development both to optimize transmission and to prevent uncontrolled parasite multiplication overwhelming the host. In this review we detail recent developments in our understanding of the molecular mechanisms that underpin the production of stumpy forms in the mammalian bloodstream and their signal perception pathways both in the mammalian bloodstream and upon entry into the tsetse fly. These discoveries are discussed in the context of conserved eukaryotic signaling and differentiation mechanisms. Further, their potential to act as targets for therapeutic strategies that disrupt parasite development either in the mammalian bloodstream or upon their transmission to tsetse flies is also discussed.

African trypanosomes, parasites that cause human sleeping sickness, undergo a density-dependent differentiation in the bloodstream of their mammalian hosts. This process is driven by a released parasite-derived factor that causes parasites to accumulate in G1 and become quiescent. This is accompanied by morphological transformation to ‘stumpy’ forms that are adapted to survival and further development when taken up in the blood meal of tsetse flies, the vector for trypanosomiasis. Although the soluble signal driving differentiation to stumpy forms is unidentified, a recent genome-wide RNAi screen identified many of the intracellular signalling and effector molecules required for the response to this signal. These resemble components of nutritional starvation and quiescence pathways in other eukaryotes, suggesting that parasite development shares similarities with the adaptive quiescence of organisms such as yeasts and Dictyostelium in response to nutritional starvation and stress. Here, the trypanosome signalling pathway is discussed in the context of these conserved pathways and the possible contributions of opposing ‘slender retainer’ and ‘stumpy inducer’ arms described. As evolutionarily highly divergent eukaryotes, the organisation and conservation of this developmental pathway can provide insight into the developmental cycle of other protozoan parasites, as well as the adaptive and programmed developmental responses of all eukaryotic cells.