Genome wide dissection of the quorum sensing signaling pathway in Trypanosoma brucei

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 transmission(1). 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.