The displacement of Syrian refugees (data from UNHCR, Syria Regional Refugee Response: Inter-agency Information Sharing Portal) [31].

<p>The displacement of Syrian refugees (data from UNHCR, Syria Regional Refugee Response: Inter-agency Information Sharing Portal) [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004545#pntd.0004545.ref031" target="_blank">31</a>].</p

Year-wise trend of CL cases reported in Middle East (from Salam et al 2014, <i>PLOS Neglected Tropical Diseases</i>) [20].

<p>Year-wise trend of CL cases reported in Middle East (from Salam et al 2014, <i>PLOS Neglected Tropical Diseases</i>) [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004545#pntd.0004545.ref020" target="_blank">20</a>].</p

Characterisation of the interaction of <i>Tb</i>PTP1 with <i>Tb</i>PIP39 (wild type and mutants), in the presence or absence of citrate using BIAcore T200.

<p><b>A</b>. Representative double reference corrected single cycle kinetic titration SPR binding curves (black), monitored on a surface of covalently stabilized His-PTP1wt, for <i>Tb</i>PIP39wt (65 µM), <i>Tb</i>PIP39 D mutant (45 µM), <i>Tb</i>PIP39 DD mutant (64 µM) or <i>Tb</i>PIP39 6364 mutant (60 µM) at 25°C in 10 mM HEPES, pH 7.4; 150 mM NaCl; 0.05% P20; 2 mM DTT; 20 mM MgCl2 in the absence of citrate. <b>B</b>. Steady state analysis of the interaction between <i>Tb</i>PTP1 and <i>Tb</i>PIP39 by surface plasmon resonance. The assay was run in 10 mM HEPES, pH 7.4; 150 mM NaCl; 0.05% P20; 2 mM DTT; 20 mM MgCl2; 2 mM citrate. A concentration dependent interaction between <i>Tb</i>PTP1wt and <i>Tb</i>PIP39wt was seen. All fits were to a 1∶1 binding model, with mass transport considerations. Kd is ∼40–50 uM. <b>C–D</b> Representative double reference corrected single cycle kinetic titration SPR binding curves (black), monitored on a surface of covalently stabilized His-PTP1wt, for <i>Tb</i>PIP39wt (C), <i>Tb</i>PIP39 D mutant (D), <i>Tb</i>PIP39 DD mutant (E) or <i>Tb</i>PIP39 6364 mutant (F) at 25°C in 10 mM HEPES, pH 7.4; 150 mM NaCl; 0.05% P20; 2 mM DTT; 20 mM MgCl₂ with 2 mM citrate. In each case, a three-fold dilution series of <i>Tb</i>PIP39 (wild type and mutants) was injected over the surface, at 30 µl.min-1 and the apparent on- and off-rate constants were by globally fitting (red) a 1∶1 kinetic binding model, with mass transport considerations, to the sensorgrams using the analysis software (v1.02, GE Healthcare) supplied with the instrument.</p

Model for the initiation of differentiation via different triggers.

<p><b>A</b>. CCA and mild acid result in an increase in <i>Tb</i>PIP39 phosphorylation that stimulates differentiation. For CCA, this is mediated via the transport of citrate/<i>cis</i>-aconitate <i>via</i> surface PAD proteins. Mild acid may inhibit <i>Tb</i>PTP1, and/or stimulate the activity of the kinase that phosphorylates <i>Tb</i>PIP39. Pronase also stimulates differentiation, independently of <i>Tb</i>PTP1/<i>Tb</i>PIP39. <b>B</b>. Mechanistically, CCA stimulates both the interaction between <i>Tb</i>PTP1 and <i>Tb</i>PIP39 (Dock), and inhibition of the stimulatory effect of <i>Tb</i>PIP39 on <i>Tb</i>PTP1 (Block).</p

Evaluation of differentiation stimuli on stumpy forms.

<p>Flow cytometry traces of EP procyclin expression on AnTat 1.1 90:13 stumpy cells. Stumpy forms (St) and Procyclic forms (Pc) were used as negative and positive controls for the EP procyclin staining, respectively (<b>A</b>) and samples from untreated stumpy cell cultures were also collected at the same timepoints as cells exposed to the different differentiation stimuli (<b>B</b>). A weak reversible cold induced expression of EP procyclin is observed. To stimulate differentiation cells were treated with 6 mM <i>cis</i>-aconitate (<b>C</b>), mild acid (<b>D</b>), pronase (<b>E</b>) and phloretin (<b>F</b>). Samples were assayed at 0 h, 1 h, 2 h, 4 h, 6 h, 24 h or 48 h after exposure to each proposed trigger. The flow cytometry traces are representative of five independent experiments, each giving similar results.</p

Differential <i>Tb</i>PIP39 phosphorylation in response to different differentiation signals.

<p>Stumpy cells were exposed to 6<i>cis</i>-aconitate (CA), mild acid (pH 5.5), pronase (4 units/ml) and phloretin (100 µM) and isolated protein samples were then reacted with a phospho-specific antibody recognising the sequence (ELDHWRTDEY*TK C) (anti-phospho <i>Tb</i>PIP39) (middle panels). The same blot was also reacted with an anti-<i>Tb</i>PIP39 polyclonal antibody (left hand panel) and with an antibody detecting trypanosome alpha tubulin as a loading control (right hand panels). Phosphorylated <i>Tb</i>PIP39 was observed with CA and mild acid treatment, but not pronase.</p

RNAi mediated ablation of <i>Tb</i>PIP39 reduces the differentiation efficiency of cells treated with cis-aconitate and mild acid, but not pronase.

<p><i>Tb</i>PIP39-RNAi stumpy cells, either uninduced (−Dox) or induced (+Dox) with doxycycline <i>in vivo</i> were harvested, inoculated into HMI-9 at 37°C before the exposure to ‘no treatment’ (<b>A</b>), 6 mM <i>cis</i>-aconitate (CA) (<b>B</b>), mild acid (pH 5.5) (<b>C</b>) or pronase (4 units/ml) (<b>D</b>). Samples of the untreated and treated (<b>B–D</b>) cultures were prepared at 0 h and 4 h and differentiation analysed by the expression of the differentiation marker EP procyclin. The treated cells showed a population of undifferentiated trypanosomes (left hand peak) representing slender cells in the population and dead or damaged cells, particularly after pronase treatment. Panel E shows histograms of the percentage of EP procyclin expressing cells at 4 h after exposure to the different treatments, the results being the mean and standard deviation of 4 experiments (3 for pronase treatment). Asterisks indicate significance (p<0.001). <i>Tb</i>PIP39 depletion resulted in reduced differentiation for the <i>cis</i>-aconitate and mild acid treated samples, but not those exposed to pronase.</p

Protease activity is not necessary for differentiation in tsetse flies.

<p><b>A</b>. Protease activity (against Chromozym Try substrate) in midgut extracts from tsetse flies fed horse serum containing <i>T. brucei</i> TSW strain trypanosomes, or <i>T. brucei</i> AnTat.1.1, either in the presence or absence of Soybean trypsin inhibitor (STI). Each bar represents the average protease activity ±SEM of 10 midguts. <b>B</b>. EP procyclin expression of stumpy trypanosomes incubated <i>in vitro</i> in HMI-9 or derived from tsetse midguts 4 h after feeding with horse serum containing the parasites, with or without soybean trypsin inhibitor (±STI). Stumpy cells were analysed at 0 hours, or after 4 hours <i>in vitro</i> in the presence or absence of <i>cis</i>-aconitate (‘CA’). To control for the chilling on ice associated with tsetse midgut dissection, some <i>in vitro</i> derived samples were also chilled for 1 hour at 4°C, particular attention being taken to precisely mimic the processing of tsetse derived samples. Scoring was carried out by immunofluorescence, with EP procyclin staining scored as negative, faint or bright in each case. In all cases samples were processed for immunofluorescence in parallel and imaged under identical settings. <b>C</b>. Representative images of trypanosomes derived from tsetse midguts 4 h after feeding either in the presence or absence of soybean trypsin inhibitor. Cells were stained with antibody to EP procyclin (Green) and DAPI (Blue). A cell categorised as ‘faint labelling is arrowed. The lower images show phase contrast images of the equivalent fields. Bar = 20 µm.</p

Phloretin is unable to induce differentiation of pleomorphic slender form cells to procyclic forms.

<p><i>T. brucei</i> AnTat 1.1 90:13 cells (8×10⁵/ml) were cultured in HMI-9 at 37°C before the addition of 6 mM <i>cis</i>-aconitate, 100 µM phloretin or the same volume of 70% ethanol as a solvent control. After culture for 48 hours at 37°C, the cells were harvested, washed in phloretin-free procyclic medium (SDM-79), then inoculated into P-79 and incubated a further 5 days at 27°C. <b>A</b>. Phloretin treatment caused growth arrest within 24 hours in the pleomorphic slender cell cultures, with no outgrowth of procyclic cells being detected after transfer to phloretin-free procyclic medium. <i>Cis</i>-aconitate treated cells differentiated and proliferated as procyclic forms under the same culture conditions. <b>B</b>. The expression of the procyclic specific marker EP procyclin was monitored by flow cytometry for each culture. Whereas most <i>cis</i>-aconitate induced pleomorphic slender cells efficiently differentiated to procyclic forms within 48 hours, and expressed high levels of EP procyclin, phloretin treated cultures remained procyclin negative.</p

<i>Tb</i>PIP39 mutants promote <i>Tb</i>PTP1 activity but lack citrate responsiveness.

<p>(<b>A</b>) Recombinant proteins of wild type and mutant <i>Tb</i>PIP39 used for biochemical and biophysical analyses. <b>(B)–(F)</b> The protein phosphatase activity of <i>Tb</i>PTP1 (0.1 µg) and various forms of <i>Tb</i>PTP1 (1 µg of wild type, and the citrate binding mutants D, DD and 6364) were each measured. The combined pNPP activity of <i>Tb</i>PTP1 and <i>Tb</i>PIP39 were assayed in the absence and presence of citrate (2 mM) or, for the wild type <i>Tb</i>PIP39, in the presence of 2 mM isocitrate, which is not a differentiation trigger (Panel F). In each case, <i>Tb</i>PTP1 pNPP activity was normalised to 1.0 and all other pNPP activities were expressed relative to that. The error bars represent SD values of 5 independent triplicate assays. Only wild type <i>Tb</i>PIP39 exhibited citrate-dependent inhibition of <i>Tb</i>PTP1/<i>Tb</i>PIP39 activity.</p