Phenotypic characteristics of <i>TbP5CDH</i> RNAi cells.

<p>A) Protein levels of TbP5CDH and TbGAPDH after three days of tetracycline-induction. Comparisons were made between non-induced (tet^-) and tet-induced (tet⁺) from both wt and ^<i>RNAi</i>TbP5CDH mutant cells. Cell lysates (30 μg of total protein per lane) were loaded and probed with antibodies as indicated before. Protein loading controls were verified by nigrosine staining of the PVDF membrane after probing with specific antibodies. B) TbP5CDH activity was determined after three days of tetracycline-induction in wt and RNAi-induced cells. Cell-free total lysates were prepared from PCF trypanosomes and used as enzyme samples. Steady-state rates were monitored spectrophotometrically (Abs_340nm) using 200 μg of each lysate to start the reaction. C-D) Growth curves of wt tet^-/+ and ^<i>RNAi</i>TbP5CDH tet^-/+ PCFs. Parasites (10⁶ cells/ml) were grown in standard SDM79 (C) or SDM79 glc^- (glucose-depleted) (D) selective media. Cell densities were determined daily and were split into fresh medium every 72h. Plots represent cumulative cell numbers determined over a period of 9 days.</p

TbP5CDH is essential for establishment of midgut infection and affects procyclin expression.

<p>A) Effect of TbP5CDH depletion in PCFs during a midgut colonization assay in tsetse flies. Teneral flies were fed with a bloodmeal that contained 5x10⁵ PCF/ml from ^<i>RNAi</i>TbP5CDH tet^-/+ cells. Bars represent the percent of trypanosome-infected flies as scored (S1-S4) by microscopy, and the sum of the each scored infection represent the total percent of infected flies per treatment (total). Number of dissected flies (n) for each group were: ^<i>RNAi</i>TbP5CDH tet^- (n = 75), ^<i>RNAi</i>TbP5CDH tet⁺ (n = 74). Differences were significant after one-way ANOVA test followed by Bonferroni test (** p<0.001). B) Immunoblotting detection of EP-procyclin in non-induced (tet^-) and RNAi-induced (tet⁺) parasites grown in SDM79 medium for five days. EP-procyclin was visualized using the anti-EP repeats mAb247 (1:2,000); anti-TcGAPDH (1:4,000) was used as control for protein loading. Expected protein sizes were 39 and ~45 kDa for TbGAPDH and EP-procyclin, respectively. C) FACS analysis of surface expression of EP-procyclin. Comparisons were made in non-induced (tet^-) and RNAi-induced (tet⁺) cells from wt and ^<i>RNAi</i>TbP5CDH parasites after four days of tet addition. Parasites were fixed (2% (v/v) paraformaldehyde and 0.05% (v/v) glutaraldehyde), incubated with mAb247 (1:500) and then labeled with mouse anti-IgG coupled to AlexaFluor488 (Invitrogen). ^<i>RNAi</i>TbP5CDH tet⁺ group exhibits two different cell populations named as EP-pop₁ and EP-pop₂. D) Intensities of mean fluorescence were determined in the cell populations obtained after mAb427 labeling. Values were calculated from four biological replicates (n = 3) and bars represent mean +SD among groups. EP-pop₁ was observed in both wt and ^<i>RNAi</i>TbP5CDH groups, whereas the EP-pop₂ population was only displayed in ^<i>RNAi</i>TbP5CDH tet⁺ cells.</p

Procyclic forms of <i>T</i>. <i>b</i>. <i>brucei</i> are auxotrophic for proline.

<p>A) Growth rates of PCF maintained in complete SDM79, SDM79 glucose-depleted (SDM79 glc^-) or SDM79 media that contained neither glucose nor proline (SDM79 glc^- pro^-). Cell densities were determined daily, and cells were split every 72h. Plots represent cumulative cell numbers determined over 9 days. B) Enzymatic assay for pyrroline-5-carboxylate reductase (P5CR) activity. Kinetic rates were determined spectrophotometrically by monitoring the NADPH oxidation (Abs_340nm) resulting from P5C reduction into proline. Activities were measured in total lysates from replicative forms of <i>T</i>. <i>brucei</i> and <i>T</i>. <i>cruzi</i> (used as positive control). The plot represents initial velocities (V₀) in the function of protein variations used in the P5CR assay. C) Detection of pyrroline-5-carboxylate synthase (P5CS) in cell-free lysates. Protein samples from replicative <i>T</i>. <i>b</i>. <i>brucei</i> and <i>T</i>. <i>cruzi</i> cells (used as positive control) were electrophoresed on SDS-PAGE, blotted onto PVDF membranes and probed with polyclonal antibodies raised against TcP5CS and Heat shock protein (HSP)-60 kDa (TcHSP60), used as reference for protein loads. Expected protein sizes were 81 and 60 kDa for TcP5CS and TcHSP60, respectively. D) Intracellular proline content in PCFs incubated under different precursors. Proline concentration was determined from cells cultivated in SDM79 media (Basal) and after one hour in PBS proline levels were depleted (PBS). Then, proline restoration was assessed over 40 min in the presence of: L-proline (L-pro) used as control, DL-pyrroline-5-carboxylate (DL-P5C/γGS), L-glutamate (L-glu), L-glutamine (L-gln), L-alanine (L-ala), L-arginine (L-arg). Reactants concentrations are detailed in the supplementary data section.</p

Proton NMR spectroscopy analysis of excreted end products from proline and [U-¹³-C]-glucose metabolism.

<p>Metabolic end products (succinate, acetate and alanine) excreted from 4 mM proline and 4 mM [U-¹³C]-glucose by the procyclic wt cell line, as well as the non-induced (tet^-) and tetracycline-induced (tet⁺) ^<i>RNAi</i>TbP5CDH mutant, were determined by proton NMR spectrometry. Each spectrum corresponds to one representative experiment from a set of five biological replicates. A part of each spectrum ranging from 1.2 ppm to 2.6 ppm is shown. Resonances corresponding to ¹³C-enriched (¹³C) and non-enriched (¹²C) succinate, acetate and alanine molecules are indicated by closed and open arrows below the spectra, respectively, and contribution of proline and [U-¹³C]-glucose to succinate, acetate and alanine is shown in the top panel by arrow heads and asterisks, respectively.</p

Effect of P5C on cell viability of procyclic forms.

<p>A) Cell viability test in PCFs incubated in poor media. ^<i>RNAi</i>TbP5CDH cells were grown for three days in SDM79 and the MTT test performed in the presence of SDM79 (considered as 100% for viability), PBS (positive control), 5 mM proline (pro), 1.5 mM DL-P5C/γGS (P5C), 5 mM glucose (glc) or 5 mM proline plus 5 mM glucose (pro+glc). B) Effect of P5C on membrane integrity of wt and ^<i>RNAi</i>TbP5CDH cells. Control and knocked-down cells were incubated by 3h with PBS added of 5 mM of L-proline (control) or 1.5 mM of DL-P5C. After this time, cells were labeled with 5 μg/ml of PI and analyzed by flow cytometry. C) Fluorescence microscopy of PCFs after 3h of P5C incubation. DNA was labeled with Hoechst probe and MitoTracker was used for mitochondrial staining, as detailed elsewhere. D) ATP content of wt, ^<i>RNAi</i>TbP5CDH tet^- and ^<i>RNAi</i>TbP5CDH tet⁺ cells after 3h of P5C incubation.</p

Scheme representing the proline-alanine cycle that occurs between <i>T</i>. <i>b</i>. <i>brucei</i> and both tsetse tissues, fat body and flight muscles.

<p>Proline combustion occurs in tsetse flight muscle (right panel), which produces alanine as the main end product. Alanine is transported to the fat body by the hemolymph (left panel). Alanine and lipids constitute the major sources for proline synthesis in the fat body. Thus, in a transamination reaction, the amino group (-NH₂) is transferred from alanine to oxoglutarate to yield glutamate and pyruvate. Pyruvate can be carboxylated to form oxaloacetate while β-oxidation of lipids becomes the main source of acetyl-CoA. The fat body TCA cycle goes from citrate to oxoglutarate, the latter which can be an acceptor of -NH₂ in a new transamination reaction to produce glutamate. This glutamate is further reduced to proline, which is then transported by the hemolymph to the flight muscles. In the flight muscles proline is oxidized to glutamate, which acts as a donor of -NH₂ in a new transamination reaction in which pyruvate is the acceptor, forming alanine and oxoglutarate [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006158#ppat.1006158.ref015" target="_blank">15</a>]. Glutamate can also be deaminated to form oxoglutarate through glutamate dehydrogenase activity. Oxoglutarate is decarboxylated and oxidized to malate through the TCA cycle. The malic enzyme converts malate into pyruvate, which can in turn be a new acceptor of -NH₂ transferred from glutamate (to form alanine and oxoglutarate, as described above) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006158#ppat.1006158.ref042" target="_blank">42</a>]. During a <i>T</i>. <i>b</i>. <i>brucei</i> infection, the parasites use part of the proline produced in the fat body and transported by the hemolymph to proliferate and colonize the MG (blue section). In <i>T</i>. <i>b</i>. <i>brucei</i>, the first steps of the proline catabolic pathway are similar to those of insect muscle cells: proline is oxidized to glutamate, which can either be converted to oxoglutarate after deamination or transaminated to produce alanine and oxoglutarate. Unlike in tsetse, oxoglutarate can be further converted into succinate, which is excreted into the extracellular medium. Succinate can also be converted into malate, which is further decarboxylated to produce pyruvate. An additional decarboxylation of pyruvate yields acetyl-coA, which can be used to produce acetate that is excreted to the extracellular medium. Succinate, acetate and alanine are the major excreted products of <i>T</i>. <i>b</i>. <i>brucei</i> insect forms resulting from proline degradative flux (dotted arrows). Alanine is excreted and could reach the hemolymph during procyclics proliferation, thus enriching the insect pool of available alanine. <i>T</i>. <i>b</i>. <i>brucei</i> also uses glutamate produced from proline for the synthesis of EP-procyclins, which are needed for midgut procyclic development. In addition, proline is critical to fuel electrons to support mt-inner membrane potential, respiratory capacity and ATP synthesis driven by OxPHOS. Midgut procyclics strictly depend on proline degradation capability for survival within tsetse fly midgut and TbP5CDH (in red) is essential for colonization and establishment of parasite infection within tsetse.</p

Assessment of mitochondrial function in PCF trypanosomes.

<p>The capacity of mitochondrial inner membrane to retain safranin dye was monitored in digitonized cells. Changes in the safranin fluorescence are representative of the mitochondrial inner membrane potential (ΔΨ_m) in PCFs. ^<i>RNAi</i>TbP5CDH cells were selectively permeabilized with digitonin until fluorescence quenching was stabilized. Then, 250 μM ADP and 50 μM calcium chloride (Ca²⁺) were added to induce depolarization of the mitochondrial (mt)-inner membrane or 0.5 μg/ml oligomycin (Omy) and 500 μM EGTA to revert it, as indicated. Finally, 0.3 μM FCCP was added to collapse the proton gradient, thus releasing the dye. B) Changes in fluorescence obtained after ADP or Ca²⁺ addition were compared between tet^-/+ cells. Statistical differences were obtained by unpaired t-test (* p<0.05). C) Oxygen consumption rates in PCFs were determined in intact cells (Basal), followed by digitonin addition to selectively permeabilize mt-inner membrane. Then, L-proline (5 mM) was added as mitochondrial substrate and respiration at state 3 was obtained after addition of ADP (250 μM). Inhibition of F_o/F₁-ATP synthase was induced by oligomycin addition, to induce the non-phosphorylating respiration (state 4). Maximum respiratory capacity (Max.) was analyzed after induction of non-coupled respiration produced by FCCP (0.3 μM). Finally, the residual oxygen consumption (Res) was determined after addition of mitochondrial inhibitor antimycin A (0.5 μM). The plot is representative of four biological replicates, and mean values were detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006158#ppat.1006158.t001" target="_blank">Table 1</a>. D) ATP levels were determined in wt tet^-/+ and ^<i>RNAi</i>TbP5CDH tet^-/+ cells grown in either SDM79 or SDM79 glc^- media for three days. ATP concentration was extrapolated from the standard curve. Bars represent mean ± SD of total ATP levels relative to parasite number. Statistical differences were determined using unpaired t-test (*** p<0.01).</p

Phenotypic characteristics of <i>TbP5CDH</i> RNAi cells.

<p>A) Protein levels of TbP5CDH and TbGAPDH after three days of tetracycline-induction. Comparisons were made between non-induced (tet^-) and tet-induced (tet⁺) from both wt and ^<i>RNAi</i>TbP5CDH mutant cells. Cell lysates (30 μg of total protein per lane) were loaded and probed with antibodies as indicated before. Protein loading controls were verified by nigrosine staining of the PVDF membrane after probing with specific antibodies. B) TbP5CDH activity was determined after three days of tetracycline-induction in wt and RNAi-induced cells. Cell-free total lysates were prepared from PCF trypanosomes and used as enzyme samples. Steady-state rates were monitored spectrophotometrically (Abs_340nm) using 200 μg of each lysate to start the reaction. C-D) Growth curves of wt tet^-/+ and ^<i>RNAi</i>TbP5CDH tet^-/+ PCFs. Parasites (10⁶ cells/ml) were grown in standard SDM79 (C) or SDM79 glc^- (glucose-depleted) (D) selective media. Cell densities were determined daily and were split into fresh medium every 72h. Plots represent cumulative cell numbers determined over a period of 9 days.</p

Analysis of TbP5CDH expression levels during the main life stages of <i>T</i>. <i>b</i>. <i>brucei</i>.

<p>A) Cell densities from both PCFs and BSFs of <i>T</i>. <i>b</i>. <i>brucei</i> were monitored during 72h of growth. Cell samples were taken at 24h and 48h, and both total-RNA and protein samples were prepared for TbP5CDH expression analysis. B) mRNA expression levels of the <i>TbP5CDH</i> were relative to the expression of <i>TbGAPDH</i>, as housekeeping gene. Bars represent mean +SD from three biological replicates (n = 3). C) Protein levels were analyzed by western blotting using anti-TcP5CDH (1:2,500) and anti-TcGAPDH (1:4,000) diluted in PBS-T plus 5% (w/v) skimmed milk. Protein relative molecular masses were 63 and 39 kDa for TbP5CDH and TbGAPDH, respectively. Protein loading controls were verified by nigrosine staining of the PVDF membrane after probing with specific antibodies. D) The mRNA levels were determined by qPCR using total RNA of <i>T</i>. <i>brucei</i>-infected fly tissues (i). Parasites were isolated from the midgut (MG), proventriculus (PV) and salivary glands (SG). Comparisons were made individually and differences were analyzed using two-way ANOVA and Tukey’s post-test. The asterisk (*) denotes the significance gene expression value (p<0.05) of PV over SG samples.</p

Respiratory capacity of ^<i>RNAi</i>TbP5CDH tet^-/+ cells.

<p>Respiratory capacity of ^<i>RNAi</i>TbP5CDH tet^-/+ cells.</p