Oligopeptide Signaling through TbGPR89 Drives Trypanosome Quorum Sensing

1G protein-coupled receptors (GPCRs) and other multipass-transmembrane proteins allow eukaryotic cells to perceive an enormous diversity of extracellular signals, enabling their response to environmental information. Conventionally, GPCRs signal through trimeric G proteins to activate intracellular signaling pathways and are an intense target of drug development for the pharmaceutical industry. However, GPCRs and their cognate signaling components are not ubiquitous throughout eukaryota, being absent in red and green algae, some chromalveolates, and most excavata. Excavates contain a wide variety of important eukaryotic microbial pathogens, including the kinetoplastida comprising Leishmania and Trypanosoma parasites. Of these, Trypanosoma brucei spp., causing human and animal trypanosomiasis, live extracellularly in the bloodstream of their mammalian host and exploit environmental information to regulate their virulence and transmissibility. Specifically, morphologically “slender form” bloodstream trypanosomes proliferate until signaled to undergo development to non-proliferative “stumpy forms” adapted for transmission. This is a quorum-sensing (QS) type response triggered by the accumulation of a “stumpy induction factor” (SIF), although the nature and mechanism of signaling remains unknown. Recently, components of the SIF response pathway were uncovered by a genome-wide RNAi screen that identified signal transduction components and gene expression regulators controlling stumpy formation. However, molecules at the cell surface that detect or transport SIF, or act at early steps in the signaling pathway, remain completely unknown, as is the SIF signal that drives QS.

2In plants, GTG1 and GTG2, members of the GPR89 protein family that are classified as orphan GPCRs, detect the extracellular phytohormone abscisic acid. In mammalian cells, GPR89 acts as an anion channel protein that is involved in Golgi pH homeostasis (GPHR, Golgi pH regulator). More recent studies suggest GPR89 family members have a location in the Golgi and ER in Dictyostelium, with mutants exhibiting developmental defects and perturbed secretory function. In each case, the GPR89 proteins have 9 transmembrane domains (TMDs), distinct from the 7 TMDs conventionally found in GPCRs, and structural bioinformatics analysis has supported the distinction of plant GTGs from the GPCR family. Representatives of GPR89 are found in each of the currently recognized supergroups, although they appear to be missing in some organisms, including certain species of fungi and the pathogenic apicomplexan Cryptosporidium.

3Here, we report the presence of a GPR89 representative, TbGPR89, in the kinetoplastid parasite, Trypanosoma brucei. This surface protein is expressed on the parasite stage that receives the QS-signal and can drive stumpy formation via the SIF signaling pathway. African trypanosomes lack conventional oligopeptide transporters, but we show that TbGPR89 can transport oligopeptides, which promote stumpy formation in vitro. Furthermore, the expression of secreted oligopeptidases by trypanosomes generates a paracrine signal to co-infecting trypanosomes, driving premature stumpy formation in vivo. Our data invoke oligopeptide signals received via TbGPR89 as the long-sought mechanism of trypanosome quorum sensing. These findings provide a novel therapeutic target for trypanosomes that is potentially refractory to the emergence and spread of resistance.

4Bioinformatic analysis of the trypanosomatid genomes identified genes encoding representatives of the GPR89 family (Figures S1A and S1B). For Trypanosoma cruzi TriTrypDB: TcCLB.508547.140, BLASTP detected similarity scores of 1.1e−16 and 2.3e−16 to A. thaliana GTG1 and GTG2, respectively, and 4.1e−10 to mammalian GPR89 (GPHR). The syntenic T. brucei gene, TriTrypDB: Tb927.8.1530, is predicted to encode 9-TMDs and a large central loop (http://wlab.ethz.ch/protter/start) (Figure 1A). All trypanosome family GPR89 family members contain a 70 amino acids GPHR_N (PFAM12537) domain with a conserved LSG motif in the N-terminal 5TM region of mammalian GPHR (http://smart.embl-heidelberg.de). An ABA-GPCR domain (PFAM12430, associated with abscisic acid binding in GTG1) is also present in most kinetoplastid GPR89 homologs (TriTrypDB: TcCLB.508547.140, E value = 8.5e−16) but is not detected in TbGPR89 of T. brucei (Figure S1C).

5An antibody targeting TbGPR89 detected expression on bloodstream slender but not on stumpy forms at the cell surface (Figures 1B and 1C). To explore the function of the protein, we transfected parasites with a plasmid driving the doxycycline-inducible ectopic-expression of TbGPR89. In T. brucei Lister 427 90:13 monomorphic cells, which have lost the capacity for stumpy formation through serial passage, the protein was effectively expressed but there was only a subtle effect on cell growth (Figure 1D). However, when the protein was inducibly expressed in developmentally competent pleomorphic trypanosomes, T. brucei EATRO 1125 AnTa1.1 90:13, the parasites underwent rapid growth arrest in G1 (Figures 1E and 1F) as the cells became morphologically stumpy (Figure 1G). This represented significantly accelerated differentiation compared to the normal differentiation kinetics of wild-type parasites (i.e., stumpy formation in 24 hr rather than >72 hr). In contrast to monomorphic parasites, the protein expression was transient, being detected 4 hr after induction but reduced at 24 hr (compare Figure 1D and 1E), consistent with the developmental loss of the protein in stumpy forms.

6To establish the physiological relevance of the TbGPR89-induced arrest in pleomorphic parasites, trypanosomes were grown in vivo. After 24 hr induction, the parasites underwent rapid growth arrest (Figure 2A), accumulating in a 1K1N cell-cycle configuration (Figure 2B) despite their low parasitemia compared to uninduced parasites. Induced parasites expressed the stumpy-specific cell marker PAD1 at day 3 post infection (Figure 2C) and, when exposed to 6 mM cis-aconitate, these cells expressed EP procyclin on 69% of cells after 6 hr, reflecting effective differentiation to the next life cycle stage, procyclic forms. This was equivalent to the stumpy forms generated at high parasitemia in uninduced parasites (Figure 2D). Thus, TbGPR89 ectopic expression activates all of the hallmarks of stumpy forms.

7Physiological stumpy formation is signaled through a soluble parasite-released “stumpy induction factor” (SIF), whose intracellular signaling pathway has been recently characterized. To establish whether the TbGPR89-mediated differentiation response was enacted through this pathway, TbGPR89 ectopic expression was induced in cell lines where RBP7, a predicted RNA binding protein necessary for SIF-induced stumpy formation, was simultaneously and constitutively silenced by RNAi. Figure 2E shows that inducible TbGPR89 ectopic-expression caused growth arrest in vitro, whereas simultaneous RBP7 RNAi prevented this, allowing the parasites to continue to grow unimpeded despite effective expression of TbGPR89. This demonstrated that TbGPR89-induced arrest is transduced through the SIF response pathway (Figure 2F).

8Attempts to deplete TbGPR89 by RNAi were unsuccessful, as was allelic replacement using drug resistance cassettes targeted to replace both TbGPR89 alleles in the trypanosome’s diploid genome. We also exploited a CreLox/thymidine kinase-based gene deletion system to inducibly delete both TbGPR89 alleles (Figure S2A). Cre recombinase induction in combination with ganciclovir selection of null mutants resulted in cell death over 5 days (Figures S2A–S2D). Thus, TbGPR89 is essential in bloodstream slender form parasites.

9Topology modeling and primary sequence analysis of TbGPR89 predicted a number of potential features: an ∼125 amino acid intracellular loop containing the PFAM12537/DUF3735 GPHR family signature, a C-terminal domain with a predicted SCF ubiquitin ligase binding phospho-degron motif (http://elm.eu.org/), and a possible N-glycosylation motif at N67 (Figure S3A). To investigate the importance of these motifs, we expressed mutant forms of TbGPR89 in which the C-terminal amino acids 415–482, containing the phosphodegron motif, were deleted (ΔC), the intracellular loop was deleted and replaced with a 6xHA tag (Δ Loop), or the potential glycosylation site was mutated (N67Q). Each was then ectopically expressed in pleomorphic trypanosomes with the endogenous TbGPR89 gene intact, and their growth and stumpy formation assessed (Figures S3B–S3E). Expression of the C-terminal truncation mutant resulted in death of the parasites within 3 hr of induction, indicating a dominant-negative effect (Figure S3B). In contrast, when the Δ Loop and N67Q mutants were ectopically expressed, the accelerated stumpy induction phenotype of wild-type TbGPR89 was lost and cells continued to proliferate (Figures S3C and S3D). To assess the N67Q mutant in more detail, we generated a Cas9 expressing T. brucei pleomorphic line (T. brucei EATRO 1125 AnTat1.1 J1339) and used CRISPR technology to replace the wild-type TbGPR89 alleles with the N67Q mutant gene (allele 1) and a hygromycin resistance gene (allele 2). Independent selected cell lines had integrated the HygR gene and the N67Q mutant allele but retained an additional wild-type gene copy, supporting the mutant being nonfunctional (Figure S3F). These cells showed increased growth in vivo compared to wild-type TbGPR89, reflecting delayed differentiation (Figure 2G).

10These results, summarized in Figure 2H, demonstrated that the accelerated differentiation phenotype generated by TbGPR89 ectopic expression was not simply a consequence of perturbation of the trafficking architecture of the cells, but rather a response resulting from the expression of a surface protein whose function was dependent on its sequence integrity. Furthermore, N67Q/WT cell line analysis supported a role for TbGPR89 in physiological SIF reception and stumpy formation.

11Assignment of TbGPR89 to the GPR89 family of proteins was based upon its overall BLAST similarity and conservation of the PFAM12537 domain. To explore tertiary conservation with this or other protein families, TbGPR89 was subjected to structural homology modeling via iTASSER (iterative threading assembly refinement) previously used to investigate predicted Arabidopsis GPCR proteins. Surprisingly, searches revealed structural similarity to voltage-gated ion channels and the POT family of proton-coupled oligopeptide transporters in the substrate recognition region (Figures 3A, S4A, and S4B). POT family transporters are present in a wide range of prokaryotes and eukaryotes and are linked to small molecule uptake. However, a conventional POT gene is missing in African trypanosomes (T. brucei, T. congolense, T. vivax) but not other kinetoplastid species (Figure 3B) leading us to hypothesize that TbGPR89 may replace POT function in these parasites. Therefore, we expressed TbGPR89 in E. coli under IPTG-inducible control and monitored the uptake of the fluorescent dipeptide β-Ala-Lys-AMCA compared to the well-characterized E. coli POT, YjdL. Figure 3C shows uptake of the dipeptidomimetic in E. coli that inducibly express TbGPR89. Supporting a transport function for TbGPR89, uptake was non-saturable up to 4 mM, increased over time, and was reduced by the proton-dependent transport inhibitor, carbonyl cyanide m-chlorophenyl hydrazone and at 4°C (Figures S4C–S4E).

12Examination of the potential substrate interacting region in TbGPR89 and Geobacillus kaustophilus POT, centered on the binding pocket of the dipeptide analog, alafosfalin positioned tyrosine 48 in TbGPR89 at a corresponding location to tyrosine 78 in the peptide-binding site of G. kaustophilus POT (Figure 3A). When TbGPR89 tyrosine 48 was mutated to histidine (Y48H mutant) and tested for β-Ala-Lys-AMCA transport capability in E. coli, uptake was reduced ∼40% (Figure 3D) despite equivalent expression of the wild-type and mutant protein (Figure 3E). This supported the oligopeptide transport function of TbGPR89.

13Having demonstrated that TbGPR89 has oligopeptide transporter activity, we explored whether a heterologous oligopeptide transporter expressed in trypanosomes could promote stumpy formation. Therefore, we expressed Ty1 epitope-tagged E. coli YjdL in pleomorphic trypanosomes under doxycycline-regulated control and observed growth arrest in vitro within 24 hr (Figure 4A). In this case, protein expression was retained over 72 hr, rather than being lost beyond 24 hr as in TbGPR89 ectopic expression (compare Figures 4B and 1E), presumably due to absence of the phospho-degron domain in the heterologous protein. Furthermore, the YjdL protein was detected at the cell surface (Figure 4C). Induction of E. coli YjdL expression also induced rapid growth arrest in vivo (Figure 4D) and the generation of morphological stumpy forms that had a characteristic branched mitochondrion (Figure 4E) and were competent for differentiation to procyclic forms (Figure 4F). Supporting a role for oligopeptide transport in the parasite response, ectopic expression of an E388A mutant of YjdL with reduced transport capability resulted in reduced growth arrest and stumpy formation compared with intact YjdL (Figure 4G).

14The demonstration that the ectopic expression of both TbGPR89 and a bacterial oligopeptide transporter in trypanosomes promote stumpy formation prompted us to explore the contribution of oligopeptide signaling to differentiation. First, pleomorphic parasites were grown in vitro in the presence of 0% to 15% brain-heart infusion (BHI) broth as a source of oligopeptides and the effects on cell proliferation (Figure 5A) and PAD1 expression (Figures 5B and 5C) assayed. In pleomorphic parasites, stumpy formation was observed in a concentration-dependent manner; at 48 hr, cells in 0% and 15% BHI supplement were 15% and 82% PAD1+ve, respectively. Monomorphic cells, which are unresponsive to SIF, showed less effect on cell growth under the same conditions (Figure 5A). Other sources of oligopeptide mixtures also provoked parasite arrest and PAD1 expression in culture, including animal peptone, tryptose, proteose peptone, and vegetable infusion (Figures 5D and 5E).

15To explore oligopeptide-mediated stumpy formation in more detail, we chemically synthesized libraries of dipeptides and tripeptides and tested their ability to accelerate stumpy formation. Specifically, combinatorial sublibraries of dipeptides or tripeptides differentiated by N-terminal specific amino acids (i.e., 16 AA-X dipeptides per sublibrary or 256 AA-X-X tripeptides per sublibrary) were evaluated, revealing specificity in the response (Figures 6A–6D). Tripeptides were more potent than dipeptides, but the tripeptides with Asn, Gln, His, Phe, Asp, and Trp at the N terminus were most effective (Figures 6C and 6D). These each arrested growth of the parasites within 48 hr and resulted in PAD1+ve cells demonstrating their effective generation of stumpy forms (Figures 6E and 6F). Correspondingly, tripeptides competed more effectively than dipeptides for β-ALA-Lys AMCA uptake in E. coli expressing TbGPR89 (Figure 6G).

16We next explored the relevance of oligopeptide signals in vivo by manipulating their generation during infections. Trypanosomes release serum-stable peptidases in vivo, some of which accumulate at high parasitemia and retain activity in blood. Examples are type I pyroglutamyl peptidase (TbPGP, TriTrypDB: Tb927.4.2670) that acts on serum substrates with an N-terminal pyroglutamyl residue (Tables S1 and S2) and prolyl oligopeptidase (TbPOP; TriTrypDB: Tb927.10.8020), which cleaves after proline residues (Tables S3 and S4). TbPGP is a cytosolic peptidase released by lysed parasites during infections whereas TbPOP is reported to be secreted. To determine if the activity of these trypanosome-derived oligopeptidases in blood could affect stumpy formation, we generated transgenic parasite lines that express TbPGP or TbPOP with a C-terminal Ty1 epitope and also modified TbPGP with a BIP N-terminal fusion (BIPN-TbPGP) promoting extracellular secretion. In vitro, the inducible expression of TbPGP and BIPN-TbPGP did not affect cell growth (Figure S5A), indicating their expression was not deleterious. In contrast, TbPOP expression slowed growth and was detectably secreted (Figures S5B and S5C). Strikingly, however, pleomorphic cells induced to express either oligopeptidase in vivo arrested and differentiated to stumpy cells at lower parasitemia than in uninduced cells. Moreover, this effect was more rapid and pronounced in parasites expressing secreted TbPGP fused with a BIPN leader than in parasites expressing the native TbPGP (Figure S6).

17We then investigated whether expression of trypanosome oligopeptidases could generate an inter-cellular paracrine signal to promote differentiation in co-infecting parasites. To test this, BIPN-TbPGP or TbPOP expressing trypanosome lines were co-infected with a distinct pleomorphic reporter cell line modified to encode a Ty1 epitope-tagged paraflagellar rod protein, allowing the visual discrimination of “producer” (peptidase secreting) and “receiver” (PFR-Ty1) cells (Figure 7A). Infections were initiated with PFR-Ty1 cells alone, or with PFR-Ty1 cells in combination with cells induced or not to express each oligopeptidase. The parasitemia of resulting infections and the proportion of “producer” (BIPN-TbPGP or TbPOP) and “receiver” (PFR-Ty1) cells was then scored, with co-labeling for PAD1 identifying the proportion of stumpy cells for each cell type (Figure 7A, right). Figures 7B and 7C show that expression of either BIPN-TbPGP or TbPOP restricted growth of the total parasite population, which grew to a much lower level than in a single infection or when peptidase expression was not induced in the co-infecting population. Despite the lower overall parasitemia, the “receiver” PFR-Ty1 cells in the oligopeptidase-induced co-infection were highly enriched for stumpy forms (Figures 7B, 7C, and S7A). Moreover, analysis of the relative proportion of “producer” and “receiver” cells in each group demonstrated that the “producer” cells were more affected than the “receiver” cells by the oligopeptidase expression, as their overall levels diminished as a contribution to the total parasitemia (Figure S7A). These observations are all consistent with the hypothesis that secreted oligopeptidases promote stumpy formation as a paracrine response in the “receiver” cells, and the producer cells are affected by their production of the peptidase in vivo, or by a combination of a local autocrine and paracrine response. We conclude, therefore, that peptidases released by trypanosomes in vivo can generate a paracrine quorum sensing signal to induce stumpy formation.

18Our data reveal oligopeptide signaling can promote trypanosome QS consistent with the activity of the proposed stumpy induction factor. This is based upon several key discoveries; (1) a GPR89 family member with oligopeptide transport capability, (2) the surface location of this protein on the parasite stage that receives the QS signal, (3) the ability of ectopically expressed TbGPR89 and a heterologous oligopeptide transporter to drive stumpy formation, (4) the capacity of specific oligopeptides to promote stumpy formation in vitro, and (5) the ability of secreted oligopeptidases, a normal part of trypanosome infection, to provide a paracrine signal promoting stumpy production in co-infecting parasites. Collectively, these provide a “signal” and “receptor” mechanism for density sensing in trypanosome infections, processes that have been the subject of speculation for over 20 years.

19In both eubacteria and eukaryotes, diverse oligopeptides are transported by promiscuous POT family proteins to provide nutritional carbon or nitrogen sources for cells, these often being generated by extracellular peptidases. POT family proteins are also present in kinetoplastids, except the African trypanosomes (T. brucei, T. congolense, T. vivax), where the POT gene has apparently been lost by gene deletion. These trypanosomes all show density-dependent growth control in the mammalian bloodstream, this being linked to the development of stumpy forms in T. brucei. We show that TbGPR89 is an essential protein in trypanosomes that can replace the oligopeptide transport function of a conventional POT but also provides a density sensing role in trypanosome quorum sensing. This dual function provides an elegant mechanism for signal perception where TbGPR89 enables essential oligopeptide uptake to the proliferating slender forms but also ensures there is a single signal input for QS without interference from additional oligopeptide uptake by a conventional POT. An accelerated developmental response to ectopic overexpression of TbGPR89 may reflect increased sensitivity to the signal, although oligopeptide transporters may also be regulated by oligomerization to precipitate differentiation. The intracellular oligopeptide specificity and response remains to be dissected, but may link to TOR and AMPK, which are regulators of trypanosome differentiation.

20Several peptidases are released by trypanosomes that accumulate and are active in the bloodstream during infections. These and additional host peptidases degrade numerous host substrates, to generate diverse oligopeptide signals that may be transported by TbGPR89. Here, we demonstrated that two released peptidases, prolyl oligopeptidase (TbPOP) and pyroglutamyl peptidase (TbPGP), increase stumpy formation. We show that TbPOP is secreted by trypanosomes, consistent with its detection in the excretory/secretory material of the parasite. Furthermore, although TbPGP is normally released by lysed trypanosomes, we observed enhanced stumpy formation when this protein is modified to enhance its secretion. That both peptidases generated the paracrine signal suggests, therefore, that common products, generated directly or after further processing in the blood, contribute to the signal.

21Oligopeptides are enriched in both trypanosome conditioned medium and the serum of infected patients. Supporting this, we show that TbPGP and TbPOP degrade serum proteins (Figure S7C; Tables S1 and S3), and addition of oligopeptide mixtures to trypanosome media promotes stumpy formation of pleomorphic trypanosomes but not monomorphic parasites that are QS-signal blind. The nature of the defect in these monomorphic cells remains to be discovered but is not at the level of TbGPR89 because this protein is essential in bloodstream form parasites.

22Both TbPGP and TbPOP exhibit activities that may be relevant for trypanosome QS. Thus, TbPGP degrades pyroglutamyl groups on serum peptides such as thyroid releasing hormone (TRH) or gonadotrophin-releasing hormone (GnRH), an activity we have confirmed in human and bovine serum (Table S1), and many additional TbPGP-generated products were detected that will require extensive further analysis for unambiguous identification (Table S2). TbPOP can also act on bioactive peptide hormones as well as abundant host collagen and proline-rich proteins in serum (Tables S3 and S4). Using synthetic di- and tripeptide libraries, we have further established that there is specificity to the oligopeptide signal with selectivity for several tripeptide sublibraries, and some members of these are detected as direct products of the secreted peptidases in serum.

23The ability of exogenous oligopeptides and secreted peptidases to induce premature differentiation in vivo supports a role for oligopeptides in inter-cellular QS. Moreover, the absence of an effect of TbGPR89 expression in developmentally incompetent parasites (monomorphs and RBP7-depleted cells), and the accelerated differentiation of a co-infecting “receiver” line demonstrates that the developmental response is not a consequence of the action of intracellular peptidase trafficking or activity at the expresser cell surface. Indeed, the expressed VSG remains intact (Figure S7B), arguing against a SIF-independent differentiation response activated by perturbation of the surface coat. Instead, our data suggest a model where released peptidases act as public goods to generate a paracrine oligopeptide signal that can promote differentiation. This is consistent with the reported properties of SIF (<500 Da, heat stable) but differs from the anticipated characteristic of SIF as a directly released metabolite or small molecule.

24A “stumpy induction factor” signal generated in the environment by the release of parasite proteases is consistent with environmental sensing in other organisms and the biological characteristics of trypanosome infection in vivo. For example, a recently reported fungal signaling system is dependent upon the release of extracellular oligopeptidase, and in Bacillus cereus, QS signaling operates by the extracellular processing of the autoinducing peptide by a secreted neutral peptidase B, and then import by an oligopeptide permease. The local production of peptidases is also compatible with the generation of stumpy forms when parasites are constrained in the host dermis or adipose tissue as well as at high density in the bloodstream circulation of infected mice. This is because both environmental flow and cell density would determine the concentration of oligopeptide signals generated, with tissue-resident parasites in a low flow environment and in close proximity to peptidase substrates predicted to differentiate at lower density than circulating parasites in a high flow blood environment. Such local effects can also explain how livestock trypanosome infections can sustain transmissibility while exhibiting low bloodstream parasitemia. Immune-mediated parasite killing could also boost the generation of transmission stages through peptidase release from dying parasites.

25Our results have implications for two potential therapeutic approaches. First, the delivery of a stable oligopeptide signal to promote premature stumpy formation could generate an anti-virulence “quorum-sensing interference” approach if comprehensively and systemically active. Alternatively, our discovery that a GPR89 family protein is required for cell viability and cell-type differentiation provides opportunities for pharmacological intervention. GPCR-like proteins as well as multi-membrane spanning transporters and transceptors are highly targeted in drug discovery programs, with nearly 40% of current drugs focused on this family of proteins. In particular, the functions of TbGPR89 in both slender form viability and parasite stumpy formation provides an evolution-proof double lock to prevent the emergence of drug-resistance, since any viable drug-resistant mutants bypassing TbGPR89 would be unable to spread through their transmission incompetence.

26Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Keith R. Matthews (keith.matthews@ed.ac.uk).

27Trypanosome infections were carried out in female healthy outbred MF1 mice at least 10 weeks old, immunocompromised with 25 mg/ml cyclophosphamide delivered intraperitoneally 24 h prior to trypanosome infection. No blinding was performed and the animals were not subject to previous procedures or drug treatment. Animal experiments were carried out according to the United Kingdom Animals (Scientific Procedures) Act under a license (PPL60/4373) issued by the United Kingdom Home Office and approved by the University of Edinburgh local ethics committee. Animals were kept in cages containing 1-5 mice on a 12h daylight cycle and maintained at room temperature. Between 2 and 3 mice were used per group; groups usually involved induction of ectopically expressed gene expression- this being activated by inclusion of doxycycline (200 μg/ml in 5% sucrose) provided in the drinking water, with control mice being provided with 5% sucrose alone. Parasitemias were estimated using a ‘rapid matching’ method involving visual comparisons of live parasites in blood by microscopy with a published standardized chart of parasite numbers per ml.

28Trypanosoma brucei EATRO 1125 AnTat1.1 90:13 (TETR T7POL NEO HYG) parasites were used throughout for all pleomorphic cell analysis. For monomorphic trypanosome analysis, T. brucei Lister 427 90:13 (TETR T7POL NEO HYG) cells were used. In vitro, parasites were grown in HMI-9 medium (Life technologies) at 37°C 5% CO2.

29Experiments were carried out without blinding or randomization/stratification. All figures include information on the replicate number for each experiment. Experiments were also validated in additional pilot experiments or independent replicates. Statistical methods were carried out as detailed under ‘QUANTIFICATION AND STATISTICAL ANALYSIS’ and no data were excluded. For the analysis of phenotypes 3 animals per treatment were routinely used for analysis. Our previous analyses (e.g., ) indicate that this sample size is sufficient to detect differences between cell lines and treatment groups. In the current manuscript, the visual analytical assays applied (manual scoring by microscope) to the different treatments and groups (cell cycle scoring, analysis of PAD1 staining, scoring of flagellar labeling, morphological analysis) required analyses to be limited to 2 animals per group in mixed infection experiments. P values of less than 0.05 were considered statistically significant.

30Parasite transfection was by Amaxa nucleofection according to previous detailed methods for monomorphic or pleomorphic parasites.

31The TbGPR89 (TriTrypDB: Tb927.8.1530), TbPGP (TriTrypDB: Tb927.4.2670) and TbPOP (TriTrypDB: Tb927.10.8020) open reading frames were amplified from T. brucei EATRO 1125 AnTat1.1 wild-type genomic DNA with appropriate primers (Table S5) with terminal restriction sites for insertion into the pDex577-Y vector for tetracycline-inducible overexpression with an C-terminal TY epitope tag. The resulting overexpression constructs were linearized with NotI and transfected into Trypanosoma brucei EATRO 1125 AnTat1.1 90:13 (Pleomorphs) or Lister 427 90:13 (monomorphs) cells. Several independent cell lines were isolated and their growth analyzed in vitro or in vivo in the presence or absence of tetracycline, or doxycycline, respectively. Expression was confirmed by western blotting using an anti-TY antibody. To generate the BIP-PGP contruct, the BIP N-terminal sequence was amplified from T. brucei genomic DNA and subcloned into the pDEX-PGPty plasmid at the N terminus with the appropriate restriction enzymes. The Bacterial YjdL gene (Escherichia coli str. K-12 substr. W3110) was amplified from BL21 genomic DNA and cloned into the pDex577-Y plasmid for integration into T. brucei pleomorphic cells. For site directed mutagenesis, the QuikChange II Site-Directed Mutagenesis Kit was used (Agilent). For generation of conditional KOs of GPR89, Trypanosoma brucei EATRO 1125 AnTat1.1 90:13 were transfected with pLEW100cre-EP1 containing the Cre recombinase. The GPR89 gene was cloned into pyrFEKO-PUR plasmid (containing 5′ and 3′ GPR89 UTRs) and transfected into T. brucei EATRO 1125 AnTAT 90-13 Cre cells. Subsequently, the second GPR89 allele was targeted using pyrFEKO-BSD. Analysis of Cre induction was carried out according to. Thus, induction of Cre with Doxycycline acts to remove both the BSD-TK cassette and the GPR89ty-Puro-TK allele, generating a null mutant. Null mutants were then selected by their sensitivity to blasticidin and puromycin, and resistance to gancyclovir (GCV), which counter-selects TK-expressing cells. 50 μg/ml GCV was used to select for the loss of TK.

32To enable the use of CRISPR tools in T. brucei pleomorphic cells, we introduced into T. brucei EATRO 1125 Antat 1.1 cells the pJ1339 plasmid (a derivative from pJ1173, gift from Dr. Jack Sunter, Oxford Brookes University, UK; unpublished) that carries a single resistance marker, puromycin, the tet repressor, T7 RNA polymerase and Cas9. Expression of Cas9 is constitutive. To replace the endogenous copy of GPR89, the N67Q mutant was cloned into pPOTv6 using HindIII and BamHI. For gene replacement with pPOTv6-GPR89-N67Q (Blasticidin) and pPOTv7 (Hygromycin) constructs, 107 cells were transfected with the PCR reactions for the two sgRNAs and two donor DNAs (combined volume approx. 100 μl) in a total volume of 250 μl.

33Methanol-fixed blood smears were rehydrated in phosphate-buffered saline (PBS) for 5 min. Slides were stained with 30 μL of 4', 6-diamidino-2-phenylindole (DAPI; 10 μg/ml in PBS) for 2 min in a humidity chamber and were then washed for 5 min in PBS. Slides were then mounted with 40 μL Mowiol containing 2.5% 1, 4-diazabicyclo(2.2.2)octane (DABCO). 250-500 cells were counted per sample and per time point except where there was very low parasitaemia, where 200 cells were counted.

342-5x106 cells were washed twice in PBS prior to fixing in 500 μL 2% formaldehyde/0.05% glutaraldehyde > 1h at 4°C. Cells were then washed 3x in PBS and resuspended in 2%BSA:PBS for 30 min. Cells were then resuspended in primary antibody diluted in 2%BSA:PBS (αEP procyclin (Cedar Lane laboratories) was diluted 1:500) and were incubated overnight at 4°C. The cells were washed twice in PBS and were resuspended in secondary antibody diluted in 2%BSA:PBS (α-mouse FITC was diluted 1:1000). The cells were washed twice in PBS and were resuspended in 500 μL PBS containing 0.02 μg/ml DAPI. Samples were then processed on an LSRII flow cytometer (BD Biosciences). Positive controls and secondary antibody only controls were included. Analysis was performed using FlowJo software (Tree Star).

35An antipeptide antibody recognizing TbGPR89 amino acids LDASQVSERIKSNFS was generated in rabbits (Eurogentec). For detection of GPR89, cells were resuspended in ice-cold 1 mM TLCK (Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride, Sigma) at 1x108 cells/ml and incubated on ice for 5 minutes then incubated 37°C for a further 15 minutes, and then diluted with to 1X with 4X 8M urea loading buffer without DTT. Protein samples were resolved on SDS-PAGE gels and blotted onto nitrocellulose membrane. Primary antibody dilutions were prepared in 1% BSA/TBS and the membrane was incubated overnight. αGPR89 antibody was used at 1:1000, αBB2 antibody was used at 1:20 to detect the TY-tagged TbGPR89, αPAD1 antibody was used at 1:1000 and αEF1 (elongation factor 1-alpha, Merck Millipore 05-235) was used for loading controls at 1:7000. Secondary antibodies were diluted in 50% TBS and 50% Li-Cor blocking buffer. Both anti-mouse (IRDye® 680 goat anti-mouse, Li-Cor) and anti-rabbit (goat anti-rabbit IgG (H+L) Dylight 800, Thermoscientific) secondary antibodies were diluted 1:7000. Signal was detected on a Li-Cor Odyssey imaging system.

36Parasites were resuspended at 2x106/ml in SDM79 media (GIBCO by Life technologies) containing 6mM cis-aconitate (Sigma, A3412) and were incubated at 27°C. Samples were collected for flow cytometry at 0h, 3h and 6h. Progression to procyclic forms was monitored by their expression of EP procyclin using flow cytometry as detailed above.

37Bloodstream-form trypanosomes (2-3x106/ml) were incubated in HMI-9-medium containing 100 nM MitoTracker Red CMXROS (Molecular Probes) for 30 min at 37°C. Then the cells were washed with HMI-9 and incubated for a further 20 min in the absence of MitoTracker, after which the parasites were fixed for 2 min at 4°C with 0.4% paraformaldehyde (prepared fresh in PBS). The cells were then washed once with PBS and air-dried smears were prepared. The slides were fixed for 10 min in methanol at 20°C, before rehydration for 10 min in PBS, followed by DAPI staining and mounting in MOWIOL.

38A single colony of E. coli BL21-CodonPlus (DE3)-RIPL cells containing the plasmids pET28a- (TbGPR89, YjdL, TbGPR89-TYR48) or empty pET28a was inoculated in 3 mL LB media containing 100 μg/mL kanamycin and 34 μg/mL chloramphenicol and allowed to grow overnight. Overnight cultures were transferred to 10 mL LB media with the same amount of antibiotics using a dilution of 1:50. The cells were allowed to grow until OD600 of 0.6–0.8 before induction with 1 mM IPTG. The cells were harvested 3 h after induction with IPTG at 37oC.

39Uptake assays were performed with bacteria 3 h after induction with IPTG with the fluorescent dipeptide β-Ala-Lys-AMCA (Biotrend, Cologne, Germany). Cells were harvested by centrifugation (2500 x g, 5 min) to an OD600 of 10 and incubated in Assay Buffer (33 mM HEPES, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4 and 5 mM glucose, pH 6.5) at room temperature for at least 20 min. In a final volume assay of 100 μl, 1.5 μL of a 20 mM β-Ala-Lys-AMCA stock solution (final concentration 500 μM) in the presence of absence of competing Di- or Tripeptide sublibraries, or with 40μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), was incubated with 40 μL bacteria cells at 37°C. Uptake was determined over 15-20 minutes. Following centrifugation and washing twice in Assay-buffer, the cell pellet was suspended in 100 μL modified Assay buffer and the uptake was quantified by fluorescence measurements (excitation at 340 nm and emission at 460 nm) on a Varioscan fluorimeter. Non-specific uptake control experiments were performed under the same conditions and procedures as described above using E. coli BL21-CodonPlus (DE3)-RIPL cells transformed with the empty pET28a vector.

40The dipeptide and tripeptide libraries were synthesized by standard Fmoc Solid Phase Peptide Synthesis via split-and-mix. Rink Amide TentaGel beads (100 mg per sublibrary, 0.22 mmol/g, 90 μm, Rapp polymer) were used for the synthesis. The amino acids used for library production were: Ala, Arg, Asn, Asp, Gln, Glu, His, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr and Val. Beads were swollen in dichloromethane for 10 min prior to coupling of Fmoc deprotection. After every synthesis step, coupling or Fmoc deprotection the beads were washed with dimethylformamide and dichloromethane. A TNBS test was performed after each step. The Fmoc amino acids (3 eq) were coupled in the presence of HATU (2.9 eq) and DIPEA (6 eq) in DMF (10 ml/mg of resin) for 20 minutes and the procedure repeated twice. The Fmoc groups were removed by shaking the beads twice for 15 minutes in a solution of 20% piperidine in DMF (10 ml/g of resin). The peptides were cleaved in a solution of 95% trifuoroacetic acid (TFA), 2.5% triisopropylsilane (TIS) and 2.5% water for 4 h. The solvent was removed in vacuo and the samples re-dissolved in water and lyophilised. The libraries were separated in sublibraries depending on the N-terminal amino acid (2 – 11 mg) were finally dissolved in dry DMSO at 500 mM concentration. All library concentrations for growth and differentiation assays were derived from the average molecular mass of the amino acids contained.

41Trypanosoma brucei EATRO 1125 AnTat1.1 90:13 parasites were incubated with varying concentrations of each sublibrary of di-or tripeptides (ranging from 500 μM to 62.5 μM) in 2 mL wells. The starting parasite density was 1x105 parasites/ml. After 48 and 72 h, cell were counted by hematocytometer and samples were taken and fixed with 4% paraformaldehyde, and assessed for PAD1 expression. Similarly, peptone broths (Brain heart infusion, Peptone from animal proteins, Proteose peptone, Vegetable Special infusion, peptone from Gelatin - Sigma-Aldrich- or Tryptose -Fluka) were prepared according to the manufacturers’ instructions, and different concentrations tested in T. brucei EATRO 1125 AnTat1.1 90:13 cultures. After 48 hr, samples were taken and assessed for PAD1 expression.

42Samples of human and bovine serum incubated overnight at 37°C in the presence or absence of TbPGP, were centrifuged for 10 minute at 13000 g and the supernatant passed through a 3000 Da cut-off centrifugal filter membrane (Amicon Ultra-0.5). The filtrate was diluted with methanol and analyzed using an Orbitrap Mass Analyzer (80-1000 Da range, positive and negative mode). Additionally, the masses detected in each sample were screened against the monoisotopic masses of pyroglutamate and a range of commonly occurring pyroglutamate-containing serum proteins. The intensity of the free pyroglutamate signal was significantly greater in all serum samples treated with TbPGPase (negative mode) while the pGlu peptides, TRH and TRH-like peptide (detected in positive mode), were present in control serum but absent from samples treated with TbPGPase (highlighted cells).

43For TbPOP, samples of human and bovine serum incubated overnight at 37°C in the presence or absence of enzyme were centrifuged for 10 minute at 13000 g and the supernatant passed through a 3000 Da cut-off centrifugal filter membrane (Amicon Ultra-0.5). The filtrate was diluted with methanol and analyzed using an Orbitrap Mass Analyzer (80-1000 Da range, positive and negative mode).

44The i-TASSER server was utilized to generate a model using the sequence of TbPOT; chain A of structure 4IKZ was specified to be used as the template. TbPOT residues equivalent to template substrate-binding residues were visually identified and prioritised for mutational analysis.

45The evolutionary history of GPR89 cDNA sequences was inferred using the Neighbor-Joining method following multiple sequence alignment using MUSCLE. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches in the generated trees. The trees are presented to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7.

46Graphical and statistical analyses were carried out in GraphPad Prism version 6 (GraphPad Software, La Jolla, California, USA, https://www.graphpad.com) by two-way repeated-measures ANOVA test followed by Bonferony post hoc analysis. For individual experiments, n values are included in the Figure legend; graphs provide mean values ± SEM.