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.

SummaryTrypanosome parasites control their virulence and spread by using quorum sensing (QS) to generate transmissible “stumpy forms” in their host bloodstream. However, the QS signal “stumpy induction factor” (SIF) and its reception mechanism are unknown. Although trypanosomes lack G protein-coupled receptor signaling, we have identified a surface GPR89-family protein that regulates stumpy formation. TbGPR89 is expressed on bloodstream “slender form” trypanosomes, which receive the SIF signal, and when ectopically expressed, TbGPR89 drives stumpy formation in a SIF-pathway-dependent process. Structural modeling of TbGPR89 predicts unexpected similarity to oligopeptide transporters (POT), and when expressed in bacteria, TbGPR89 transports oligopeptides. Conversely, expression of an E. coli POT in trypanosomes drives parasite differentiation, and oligopeptides promote stumpy formation in vitro. Furthermore, the expression of secreted trypanosome oligopeptidases generates a paracrine signal that accelerates stumpy formation in vivo. Peptidase-generated oligopeptide QS signals being received through TbGPR89 provides a mechanism for both trypanosome SIF production and reception.

Quorum sensing (QS) is commonly used in microbial communities and some unicellular parasites to coordinate group behaviours1,2. An example is Trypanosoma brucei that causes Human African trypanosomiasis, as well as the livestock disease, nagana. Trypanosomes are spread by tsetse flies, transmission being enabled by cell-cycle arrested ‘stumpy forms’ that are generated in a density-dependent manner in mammalian blood. QS is mediated through a small (<500 Da), non-proteinaceous, stable but unidentified ‘stumpy induction factor’3, whose signal response pathway has been identified. Although QS is characterised in T. brucei, co-infections with other trypanosome species (T. congolense, T. vivax) are common in animals, generating the potential for interspecies interactions. Here, we show that T. congolense exhibits density-dependent growth control in vivo and conserves QS-regulatory genes, of which one can complement a T. brucei QS signal-blind mutant to restore stumpy formation. Thereafter we demonstrate that T. congolense-conditioned culture medium promotes T. brucei stumpy formation in vitro, dependent upon integrity of the QS signalling pathway. Finally, we show that, in vivo, co-infection with T. congolense accelerates differentiation to stumpy forms in T. brucei, this also being QS dependent. These cross-species interactions have important implications for trypanosome virulence, transmission, competition and evolution in the field.