Abstract
The protozoan parasites Trypanosoma brucei spp. cause important human and livestock diseases in sub-Saharan Africa. In 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 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 signalling 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 RNA interference library screen to identify the signalling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) or 8-pCPT-2′-O-methyl-5′-AMP to select cells that were unresponsive to these signals and hence remained proliferative. Genome-wide Ion Torrent based RNAi target sequencing identified cohorts of genes implicated in each step of the signalling 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. The putative RNA-binding protein, RBP7, was required for normal quorum sensing and promoted cell-cycle arrest and transmission competence when overexpressed. This study reveals that quorum sensing signalling in trypanosomes shares similarities to fundamental quiescence pathways in eukaryotic cells, its components providing targets for quorum-sensing interference-based therapeutics.
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Acknowledgements
This work was funded by a Wellcome Trust Programme grant (088293MA) to K.M. and by a Wellcome Trust strategic award (095831MA) to the Centre for Immunity, Infection and Evolution. D.H. is supported by a Wellcome Trust senior investigator award (100320/Z/12/Z). We thank M. Chase-Topping for statistical support.
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Conceived the study (K.M.), developed and carried out the screen (P.M., A.C., D.H., K.M.), cloned and analysed the screen outputs (B.M.M., P.M., A.C., A.I., D.H., K.M.), derived RNAi lines (B.M.M.) and analysed these in vitro (B.M.M.) and in vivo (B.M.M., J.Y.). Genomic analyses were performed by A.I.; F.R. optimized culture methods for pleomorphic cells and performed MitoTracker assays. Northern blots were performed by P.M., cell-cycle scoring and analysis was carried out by B.M.M. The paper was written by K.M., B.M.M., P.M. and A.I.
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Extended data figures and tables
Extended Data Figure 1 RNAi insert analysis after selection with pCPT-cAMP or 8-pCPT-2′-O-Me-5′-AMP.
a, RNAi target amplicon profiles from parasite lines either before (BS) or after selection with 100 µM pCPT-cAMP (left panel) or 10 µM 8-pCPT-2′-O-Me-5′-AMP (right panel). Genomic DNA was isolated from each parasite population and amplified with the Lib2F and Lib2R primers flanking the target insert site in the RNAi vector used to generate the RNAi library20. Independently selected parasite populations amplified similar products within each selection regimen, although there was more heterogeneity in band intensity when selecting with 8-pCPT-2′-O-Me-5′-AMP. Individual populations were then analysed by Ion Torrent sequencing, population 2 being used for the pCPT-cAMP selection and population 5 being used for the 8-pCPT-2′-O-Me-5′-AMP selection, these being most representative of the pool of amplicons across all of the populations in each selection. b, Ion Torrent sequence reads for the genes analysed in Figs 2 and 3 of this manuscript. In each case more than one RNAi fragment targets the genes of interest validating their selection in the screen. The pictograms of Ion Torrent read-alignments for all screen hits are included in Supplementary Data Set 2. The Ion Torrent reads derived from the 8-pCPT-2′-O-Me-5′-AMP (‘AMP’, light pink) and pCPT-cAMP (‘cAMP’, light blue) selected populations are shown for each, with the primer binding sites highlighted (in red and blue).
Extended Data Figure 2 In vitro analysis of selected targets for resistance to pCPT-cAMP.
a,The purine salvage pathway in trypanosomes, identifying those genes (in red) that were selected in either selection regimen and those (in blue) that were not. The predicted overall effect of the selected targets is to reduce AMP levels within the cell. b, In vitro growth of independent pleomorphic RNAi lines generated to validate the genes selected in each genome wide screen. In each case data represent the mean ± s.e.m. of three replicate growth assays. For each assay, cells were grown with or without tetracycline, to induce RNAi, and with or without 100 µM pCPT-cAMP. Validation is expected to provide better growth in the presence of pCPT-cAMP when RNAi is induced. RNAi induction may or may not reduce growth depending on whether gene silencing for the target gene is deleterious, this potentially complicating interpretation of pCPT-cAMP resistance. For PP1 RNAi there is growth inhibition, although growth is somewhat elevated over growth in pCPT-cAMP without RNAi induction. NEK1, YAK, RBP7, HYP2 and DS-PHOS show relative resistance to pCPT-cAMP upon RNAi induction. HYP1 shows little difference between induced and uninduced samples, RNAi being leaky in this case (see Extended Data Fig. 4b). c, In vitro growth of monomorphic and pleomorphic RNAi lines targeting ADSL and ADSS generated to validate the genes selected in each genome wide screen. For each gene RNAi causes a strong growth inhibition that is somewhat rescued by provision of the cell permeable pCPT-cAMP (for ADSS in monomorphs and ADSL in pleomorphs), indicating that purine levels are restored to aid survival/growth in these cases.
Extended Data Figure 3 In vivo analysis of ADSL and ADSS RNAi.
In vivo growth of pleomorphic RNAi lines targeting ADSL and ADSS. RNAi was induced by provision of doxycycline to the infected animals (red lines), with parallel infections remaining uninduced. A strong reduction in growth was observed in each of the cell lines, although RNAi resistant ADSL cells grew out afer 6 days, this resulting in an increase in the abundance of the transcript in the induced outgrowing population with respect to the uninduced population. Uninduced cells showed reduced ADSL transcript compared to AnTat1.1 cells, indicating that the RNAi was leaky despite the depletion being not sufficient to prevent growth. Loading on the northern blot is indicated by ethidium bromide stained rRNAs.
Extended Data Figure 4 Morphological and RNA-depletion analysis of stumpy formation regulators in vivo.
a, Morphology of cells induced, or not, to deplete NEK, RBP7A/B, HYP1, DS-PHOS and YAK. In each case the induced cells remain predominantly slender in morphology. For RBP7 and HYP1, the uninduced cells are also largely slender since RNAi is leaky in these lines. Parallel infection with AnTat1.1 generated >80% intermediate and stumpy forms at the same time point (day 6 post infection) (these being shown in Fig. 2b). Bar, 12 µm. b, Northern blots of the RNA from AnTat1.1 90:13 cells and from several of the RNAi lines grown in vivo, with or without doxycycline. For the RBP7 RNAi line and the HYP1 RNAi line the RBP7A/B transcripts (which are not distinguishable) and HYP1 transcripts are significantly depleted whether doxycycline is present or not, demonstrating leaky RNAi and explaining the loss of stumpy formation in both induced and uninduced populations observed in Figs 2 and 3. For the NEK targets, several transcripts are detected (representing Tb927.10.5930/40/50), each being depleted upon induction of RNAi, confirming cross RNAi targeting of these genes. The HYP2 transcript is detected as a smear on distinct northern blots, but this is significantly reduced upon RNAi induction. YAK RNA is detected as two bands (the upper one being the predicted size of the intact transcript) both of which are reduced upon RNAi induction. In each case ethidium bromide stained rRNA provided a control for equivalent loading.
Extended Data Figure 5 Developmental phenotypes generated by PP1 RNAi in pleomorphic trypanosomes grown in mice.
a, Flow cytometry of PAD1 expression in PP1 RNAi cells (each grown in mice ± doxycycline) at day 6 post infection. Slender and stumpy cells provide negative and positive controls for PAD expression, respectively. As cells differentiating to procyclic forms also express PAD1, the parasites were counterstained for EP procyclin expression (upper quartiles) to confirm their bloodstream cell status. b, PP1 RNAi cells retain a slender cell mitochondrial morphology. MitoTracker Red analysis of the mitochondrial elaboration of cells induced, or not, to deplete PP1 by RNAi. Cells were collected on day 5 post infection for induced and uninduced populations and the cells analysed using MitoTracker red as described in ref. 39. The uninduced cells show a branched, elaborated mitochondrial morphology typical of stumpy forms, whereas the induced cells show a less elaborated linear mitochondrial staining typical of slender cells. Example slender and stumpy control cells are shown below the panel. Bar, 12 µm. c, Flow cytometry of EP-procyclin expression for AnTat1.1 and PP1 RNAi cells grown in the presence or absence of doxycycline. Trypanosomes were collected on day 6 post-infection, then incubated with 6 mM cis-aconitate (CCA). At 6 h after CCA addition PP1 depleted cells show significantly reduced ability to differentiate to procyclic forms (n = 3; general linear model, F1,4 = 10.87, P = 0.030).
Extended Data Figure 6 PP1 RNAi cells differentiate less efficiently to procyclic forms than uninduced cells.
a, Cells, induced or not to deplete PP1 by RNAi, collected on day 6 post infection and then incubated for 6 h in SDM79 culture medium in the presence of 6 mM cis-aconitate. The uninduced samples, which retain the ability to form stumpy cells, express procyclin whereas the induced cells, which remain slender, do not. The nucleus and kinetoplast DNA are stained with DAPI. Bar, 16 µm. b, Kinetoplast-posterior dimension of cells after 24 h in differentiation conditions. PP1 RNAi-induced cells (‘plus DOX’) have not repositioned their kinetoplast whereas PP1 uninduced cells (‘no DOX’) have undergone the normal kinetoplast repositioning associated with the differentiation of pleomorphic parasites to procyclic forms40. Horizontal bars represent the median distance in each population; y axis, microns. Example cells are shown below the graph; bar, 16 µm.
Extended Data Figure 7 Growth of pleomorphic cells induced, or not, to overexpress RBP7B.
a, T. brucei AnTat1.1 90:13 cells were transfected to enable tetracycline-inducible ectopic expression of RBP7B (Tb927.10.12100) from the pDEX-577-Y construct36. No epitope tag was incorporated into the encoded protein to prevent tag-induced disruption of protein function. b, Three clones were isolated; when RBP7B expression was induced the cells exhibited strong growth inhibition in vitro approximately 48 h post induction. Clone 2 was used for the subsequent in vivo analyses (Fig. 4a–d). Analysis of monomorphic cells engineered to overexpress RBP7B did not demonstrate any growth reduction, although ectopic expression of the RBP7B transgene was not confirmed by northern blotting (data not shown). c–f, In vivo growth of pleomorphic cells induced, or not, to overexpress RBP7B. The cell-cycle types of cells on different days of infection either uninduced to overexpress RBP7B, or with induction started on day 0 of infection or on day 3 of infection are shown. c, Infections initiated with the RBP7B overexpression line in the absence of doxycycline induction. d, For parasites where RBP7B is induced on day 0 of infection, the cells show accelerated accumulation of 1K1N (G1) cells with >95% cells being 1K1N at day 3, compared with <90% for the uninduced cells (c). e, No differential growth inhibition was observed when RBP7B expression was induced on day 3 of infection as the cells were already undergoing cell-cycle arrest and stumpy formation from day 4 onwards in control and treated samples. f, The day 0 induced cells grew more slowly during the course of infection, though complete arrest of the population was not observed. g. Morphology of cells on day 3 post infection where RBP7B overexpression is induced (+DOX) or not (–DOX) from day 0. Morphologically stumpy (St) and intermediate (Int) cells were present in the induced population, albeit at low frequency (typically approximately 15% by manual scoring) but not the uninduced population. Bar, 16 µm.
Extended Data Figure 8 Consequences of RBP7B overexpression or RBP7A/B depletion on the T. brucei transcriptome.
a, Experimental schematic for the identification of transcripts regulated upon RBP7 ablation by RNAi or overexpression. In each case RNAi or overexpression (OE) cell lines were inoculated in four mice, providing two bioreplicates for ‘plus DOX’ and ‘minus DOX’, respectively, for each sample. A parallel infection of the parental T. brucei AnTat1.1 90:13 line was also performed. At three days post-infection (when infections were all at approximately equivalent parasitaemia, though RBP7B OE reduced this, as shown in Fig. 4b) the parasites were collected, purified by DEAE chromatography and RNA prepared. This was then subject to RNA-Seq analysis at BGI Tech Solutions and transcript profiles analysed as detailed in the Methods. Since RBP7 RNAi was leaky, comparisons analysed both the DOX induced and uninduced samples versus the equivalent AnTat1.1 samples, rather than comparing the ‘plus DOX’ and ‘minus DOX’ samples. b, Scatterplot of the expression profile from RBP7B overexpressing cells induced with doxycycline, or not. The positions of RBP7 and procyclin transcripts are indicated to the left of each spot. c, Scatterplot of the expression profile from RBP7 RNAi cells with and without doxycycline versus AnTat 90:13 cells with and without doxycycline. Major downregulated transcripts are indicated to the right of each spot.
Extended Data Figure 9 Transcripts regulated upon perturbed RBP7 expression in pleomorphic T. brucei.
a, Scatterplots of the expression profile of genes from RBP7 RNAi (± DOX combined) cells versus RBP7B overexpressing cells induced with doxycycline. The full comparisons are available in Supplementary Data 4. The positions of several major transcripts up or downregulated in the respective comparisons are indicated to the left and right of each spot depending on whether they are more highly expressed in the RBP7 RNAi line or the overexpressing line, respectively. b, Expression profile of the top 20 genes that show opposing regulation upon RBP7 depletion by RNAi or overexpression. Reads map to both RBP7A and RBP7B such that they are not accurately distinguished in whole-transcriptome analysis, although specific analysis of these genes confirmed overexpression of RBP7B only (data not shown). Interestingly, similar specific analysis of PAD1 and PAD2 expression revealed that they were not detectably regulated by RBP7 overexpression or RNAi.
Extended Data Figure 10 In silico analysis of the quorum-sensing gene-set.
30 non-redundant genes identified by RNAi target sequencing (RIT-seq). a, The heat map shows fitness scores (red indicates loss-of-fitness) from a prior analysis following knock-down in bloodstream form cells (blood), in insect-stage cells or during differentiation (DIF)41. Groups of predicted proteins with related functions are indicated. b, The quorum sensing gene-set displayed significant (P = 0.0008, χ2 test) and specific enrichment in the set of (545) genes previously associated with loss-of-fitness during differentiation. c, Gene Ontology terms enriched within the quorum sensing gene-set are shown (http://genome.crg.es/GOToolBox/).
Supplementary information
Supplementary Information
This file contains Supplementary Tables 1-3. (PDF 319 kb)
Supplementary Data
This zipped file contains Supplementary Data Files 1-4, which are as follows: (1) Genomic location of Ion TorrentTM reads derived from PCR amplicons analysed from selection with either 100µM pCPTcAMP or 10M 8-pCPT-2’-O-Me-5’-AMP. Those with Lib2F or Lib2R primers were at the boundaries of each amplicon, whereas internal primers represent fragments internal to the amplicon boundaries. File format: Microsoft excel: ‘supplementary datafile 1.xls’. The dataset is deposited at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=drwzvqawmowsgdu&acc=GSE46501; (2) Pictograms representing the ion torrent read density for individual genes analysed or described in this study. 8-pCPT-2’-O-Me-5’-AMP selections are presented in the upper panel (pink), pCPTcAMP selections (pale blue) in the lower panel. The Lib2F and Lib2R primers are highlighted in a distinct colour (deep blue and deep red) to the internal reads. File format: png; (3) Interproscan (http://www.ebi.ac.uk/Tools/pfa/iprscan/) profiles of identified components from the screen for drivers of stumpy formation. Hits targeting purine metabolism are not shown. File: ‘supplementary datafile 3.pdf’; (4) Expression comparisons between parasites induced to deplete or overexpress RBP7. The comparison shows cells induced to deplete RBP7 by RNAi or overexpress RBP7 as an ectopic copy. File format: Microsoft excel. Files: ‘RBP7 OE plus vs minus dox.xls’; ‘RBP7 RNAi vs. OE.xls’;’ RBP7 RNAi±dox vs AnTat±dox.xls’.This analysis, plus transcriptome analyses of samples comparing RNAi lines ±doxycycline versus AnTat1.1 90:13 cells ±dox, and RBP7 overexpressing lines ‘plus dox’ versus ‘minus dox’ are deposited at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=drwzvqawmowsgdu&acc=GSE46501. (ZIP 9530 kb)
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Mony, B., MacGregor, P., Ivens, A. et al. Genome-wide dissection of the quorum sensing signalling pathway in Trypanosoma brucei. Nature 505, 681–685 (2014). https://doi.org/10.1038/nature12864
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DOI: https://doi.org/10.1038/nature12864
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