Genome-wide dissection of the quorum sensing signalling pathway in Trypanosoma brucei

Abstract

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.

Trypanosoma bruceimetabolite indolepyruvate decreases HIF-1α and glycolysis in macrophages as a mechanism of innate immune evasion

“…These parasites are auxotropic for purines and production of adenine nucleotides from hypoxanthine, the purine source in the medium, requires aspartate during the synthesis of the adenylosuccinate precursor. Indeed the enzyme responsible, adenylosuccinate synthase, is also essential (42). It is noteworthy that the rate of succinate production from glucose (which can be taken as a measure of oxaoacetate production via PEPCK) by bloodstream forms, is 201 nmol·h −1 ·mg −1 , which is equivalent to ∼56 nmols/h/5 × 10 7 cells (41), whereas the rate of excretion of indolepyruvate ranges from ∼20-50 nmols/h/5 × 10 7 cells depending on the extracellular concentration of tryptophan.…”

Cancer in the parasitic protozoans Trypanosoma brucei and Toxoplasma gondii

“…In metazoan cells, there is significant intercellular communication that directs the differentiation status of a cell, and, of course, such interactive control would not be expected to occur between independent organisms. However, as will be seen later, "quorum sensing"-the communication between single-cell organisms-has recently been described in detail in the single-cell blood parasite Trypanosoma brucei (29). In any case, protozoan parasites resemble normal functioning cells in terms of cell multiplication and differentiation from one life cycle stage to another, which are necessitated for successful growth and survival in the metazoan hosts and vectors (30).…”

Trypanosoma brucei metabolism is under circadian control

“…Slender-forms differentiate into the transmissible stumpy-form, via a mechanism of quorum sensing typically triggered in vitro at densities higher than ~10 6 parasites/mL 11 . These two life cycle stages are transcriptionally and metabolically different, which could add noise to our circadian studies 12,13 .…”