Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense
© Yamasaki et al. 2016
Received: 30 October 2015
Accepted: 3 May 2016
Published: 23 May 2016
Since Trypanosoma spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life-cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglobin (Hb) receptor. The gene was initially reported to encode a T. congolense haptoglobin (Hp)-Hb complex receptor (TcHpHbR) based on its similarity to a gene encoding a T. brucei Hp-Hb complex receptor (TbHpHbR).
Trypanosoma congolense IL3000 was used in this study. A TcHpHbR gene was PCR amplified from the parasite genome. The recombinant protein was used as an immunogen to raise antibodies for immunofluorescence assay and immunoblotting. Hemoglobin uptake by the parasite was examined by using Alexa 488 labelled Hb and visualized by confocal laser scanning microscopy. The qualitative and quantitative interaction between TcHpHbR and its ligand were measured using a surface plasmon resonance assay.
We found that, unlike TbHpHbR, TcHpHbR was exclusively expressed in the EMF stage at RNA and protein levels. The recombinant TcHpHbR (rTcHpHbR) was co-precipitated with free-Hb in a GST-pull down assay. Surface plasmon resonance revealed that rTcHpHbR binds free-Hb with high affinity (dissociation constant (K d) = 2.1×10-8 M) but free-Hp with low affinity (K d = 2.2×10-7 M). Furthermore, Alexa 488-labelled-Hb was only taken up by the EMF and co-localized with tomato lectin, which is a marker of endocytic compartments (flagellar pocket and lysosome).
We conclude that the T. congolense EMF takes up free-Hb via TcHpHbR, a receptor which is specific to this developmental stage. We therefore propose renaming TcHpHbR as T. congolense EMF-specific Hb receptor (TcEpHbR).
Many living organisms consume oxygen for energy production. Heme proteins are greatly involved in the metabolism of oxygen. Since heme proteins have essential roles in biological activities such as respiration, antioxidation and drug metabolism , eukaryotes are generally capable of de novo heme synthesis. Heme is synthesized from succinyl Co-A and glycine through eight catalytic steps and incorporated into heme proteins such as cytochrome c and peroxidase . However, previous studies and a whole genome analysis revealed that trypanosomatids, such as Trypanosoma spp. and Leishmania spp. lack key enzymes for heme biosynthesis. The parasites therefore depend on their host as a source of heme. Hemoglobin and heme uptake have been studied in T. cruzi, T. brucei and Leishmania spp. Trypanosoma cruzi possesses an ATP binding cassette (ABC) transporter for hemoglobin uptake, whereas Leishmania spp. have one hemoglobin receptor and an ABC transporter [3–5]. Trypanosoma brucei, on the other hand, possesses a haptoglobin (Hp)-hemoglobin (Hb) complex receptor (TbHpHbR, Gene ID: Tb927.6.440), which is exclusively expressed in the blood stream form (BSF) of the parasite . In mammalian blood, hemoglobin, which is released through hemolysis, binds to haptoglobin to form a complex which is immediately detoxified and taken up by macrophages for hemoglobin metabolism . Thus, T. brucei BSF takes up the Hp-Hb complex via TbHpHbR-mediated endocytosis [6, 8, 9]. It was reported that T. congolense, a causative agent of nagana, which is a devastating disease of domesticated and wild animals in Africa, possessed an orthologue of the TbHpHbR gene (Gene ID: TcIL3000.10.2930, TcHpHbR), and the crystal structure of TcHpHbR protein was revealed . Interestingly, an exhaustive proteome analysis suggested that, unlike TbHpHbR, TcHpHbR appeared to be exclusively expressed in the epimastigote form (EMF) of T. congolense . The vector stages of trypanosomes, particularly the procyclic form (PCF) and EMF, appear to require a greater amount of heme than the BSF due to their fully activated cytochrome-mediated mitochondrial respiration [11, 12]. However, the mechanisms underlying heme or hemoglobin uptake in the vector stages of African trypanosomes remain to be elucidated. The tsetse fly (Glossina spp.), which is the sole vector of African trypanosomes, periodically ingests blood meals from mammalian hosts. Thus, it is expected that the vector stages of the parasite will be exposed to a high concentration of free-Hb in each blood meal of the tsetse fly. Based on the proteomic data showing that this molecule was expressed only in EMF of T. congolense , we therefore hypothesized that the TcHpHbR would be the EMF-specific hemoglobin receptor of the parasite. As T. congolense IL3000 can be grown in all of the four main life-cycle stages in vitro, we utilized this cell line to elucidate the developmental expression of TcHpHbR. Recombinant TcHpHbR (rTcHpHbR) was used to determine the ligand specificity of the receptor [13–15].
Trypanosomes and culture conditions
Trypanosoma congolense IL 3000 (TcIL3000) strain, which was isolated on the border of Kenya and Tanzania and T. brucei brucei GUTat 3.1 strain, which was isolated in Uganda, were used in the present study and were cultured as previously described . Briefly, T. congolense and T. brucei BSFs were cultured in HMI-9 medium supplemented with 20 % fetal bovine serum (FBS) at 33 °C or 37 °C, respectively . T. congolense PCFs and EMFs were cultured in TVM-1 medium containing 20 % FBS at 27 °C. T. congolense metacyclic form (MCF) were prepared from the supernatant of the medium, in which the T. congolense EMFs were grown, and purified using DE 52 anion-exchange column chromatography .
Production of TcHpHbR and TbHpHbR proteins
The primers used in the present study
18s rRNA F
18s rRNA R
Five female 7-week-old ICR mice (CLEA Japan, Inc., Tokyo, Japan) were immunized with 50 μg (50 μl in volume) of His-tagged rTcHpHbR, which was emulsified in an equal volume of adjuvant TITERMAX® GOLD (TiterMax USA Inc., Norcross, USA). The immunizations were performed by subcutaneous injection (one primary and four booster injections) at two week intervals. Two weeks after the last booster injection, blood was collected by cardiac puncture at terminal anesthesia. Serum was prepared by centrifugation of coagulated blood at 15,000 × g for 1 min at room temperature. The animal experiments were performed in accordance with the standards of animal experimentations in Obihiro University of Agriculture and Veterinary Medicine (No. 27–92).
Southern blot analysis
Genomic DNA extracted from TcIL3000 was digested with restriction enzymes Nsi I, Sac II and Pst I (Roche Diagnostics K. K., Tokyo, Japan). After restriction enzyme digestion, DNA (10 μg/well) was separated in a 1 % agarose gel. The electrophoretically separated DNA was transferred onto a nylon membrane (Cat. No. RPN303B, GE Healthcare Bio-Sciences, UK) and the membrane was hybridized with alkaline phosphatase-labelled DNA probes (Cat. No. RPN3680 Alkphos Direct Labeling Reagent, GE Healthcare Bio-Sciences). The DNA probes for the detection of the TcHpHbR gene were prepared by a PCR using primers shown in Table 1. For visualization, the membrane was incubated in CDP-STAR detection reagent (Cat. No. RPN3682, GE Healthcare Bio-Sciences).
Northern blot analysis
Total RNA from TcIL3000 BSF, PCF, EMF and MCF was extracted using RNA extraction reagent (Cat. No. 15596–018, Thermo Fisher Scientific, Hudson, USA) according to the manufacturer’s instructions. Ten micrograms of total parasite RNA was separated on a 0.8 % agarose gel containing 2.2 M formaldehyde in 3-[N-morpholino] propanesulfonic acid (MOPS) buffer. The RNA was transferred onto a nylon membrane (GE Healthcare Bio-Sciences Corp.) and then fixed to the membrane by UV-induced crosslinking. The transferred RNA was probed with alkaline phosphatase-labelled DNA probes (GE Healthcare Bio-Sciences Corp.) under high-stringency conditions. The DNA probes to detect TcHpHbR mRNA and the reference transcript (18S ribosomal RNA) were prepared by a PCR using the primers shown in Table 1 . Probe binding was visualized with CDP-STAR detection reagent (GE Healthcare Bio-Sciences Corp.) according to the manufacturer’s instructions.
Total proteins were extracted from parasites by incubating them in cell lysis buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10 % glycerol, 1 % Triton-X 100, protease inhibitor cocktail (Cat. No. 1836153, Roche Diagnostics K. K.)) for 4 h at 4 °C. The protein extracts (2 μg) were separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrophoretically transferred onto a polyvinylidene difluoride membrane (Cat. No. RPN303F, GE Healthcare Bio-Sciences Corp.). Western blotting was performed as previously described .
Confocal laser scanning microscopy
BSF, PCF, EMF and MCF cells were collected from culture supernatants, and washed 3 times with PBS. The cell suspensions were spread over glass slides, air-dried and fixed with methanol. The specimens were incubated with 1:100-diluted anti-rTcHpHbR mouse sera and 20 μg/ml biotinylated tomato lectin (Cat. No. B1175-1, Vector Laboratories, Burlingame, USA). After washing with PBS containing 0.05 % Tween 20 (PBS-T), the slides were incubated with 1:200-diluted anti-mouse IgG conjugated with FITC (Wako Pure Chem., Osaka, Japan) and 30 μg/ml streptavidin conjugated with fluorochrome (Cat. No. SA-5594, Vector laboratories). Nucleus and kinetoplast DNA was stained with Hoechst 33342 (Cat. No. 346–07591, Dojindo, Kumamoto, Japan). A confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany) was used to observe the prepared specimens.
Hemoglobin and haptoglobin uptake
Red blood cells from defibrinated bovine blood were washed 3 times in PBS by centrifugation at 910 × g for 7 min. The red blood cells were then resuspended in PBS, diluted 10 times with sterilized distilled water to induce hemolysis and centrifuged at 15,000 × g for 10 min at 4 °C. In order to obtain bovine Hb powder, the supernatant was lyophilized using a freeze-drier (VD-500R, TAITEC, Saitama, Japan), and kept at -30 °C until use. Bovine Hb and commercially purchased Hp (Cat. No. 8010, Life Diagnostics Inc., West Chester, USA) were labelled with Alexa 488 (Hb A488 and HpA488) (Cat. No. A-10235, Thermo Fisher Scientific). HpA488 Hb complex was prepared by mixing an equal volume of 2 mg/ml HpA488 and 2 mg/ml Hb solution for 30 min at 37 °C. TcIL3000 PCF, EMF and MCF were incubated in TVM-1 medium with 20 μg/mL HbA488, HpA488 or HpA488 Hb complex for 2.5 h at 27 °C, while TcIL3000 BSF parasites were incubated in HMI-9 medium with 20 μg/ml HbA488, HpA488 or HpA488 Hb complex for 2.5 h at 33 °C. Thereafter, the parasites were placed on glass slides, air-dried and fixed with 100 % methanol for 10 min at room temperature. Nuclei and kinetoplasts were stained with Hoechst 33342 for 30 min at 37 °C. The fixed parasites were incubated with PBS containing 20 μg/ml biotinylated tomato lectin for 1 h at 37 °C. The parasites were then washed with PBS-T 3 times and incubated with PBS containing 30 μg/ml fluorochrome-labelled streptavidin for 1 h at 37 °C. A confocal laser scanning microscope (TCS-NT, Leica Microsystems GmbH) was used to observe the prepared specimens.
GST pull-down assay
To analyze the interaction between TcHpHbR and its ligand, a GST pull-down assay was performed using GST fusion rTcHpHbR (GST-rTcHpHbR) and glutathione Sepharose beads 4B . The beads and GST-rTcHpHbR were incubated with FBS diluted twice with PBS, or 2 mg/mL Hb in PBS overnight at 4 °C. After washing, the bound proteins were eluted with SDS sample buffer (125 mM Tris pH 6.8, 10 % 2-mercaptoethanol, 4 % sodium dodecyl sulfate, 10 % sucrose, 0.01 % bromophenol blue). Finally, the eluted samples were separated by 15 % SDS-PAGE and stained with Coomassie Brilliant Blue.
Surface plasmon resonance assay
Qualitative and quantitative interactions between TcHpHbR and its ligand were measured using a surface plasmon resonance assay (SPR). SPR was performed using a Biacore X analytical system (GE healthcare Bio-Sciences Corp.). The qualitative interaction between the His-tagged recombinant proteins (rTcHpHbR, rTbHpHbR) and the analytes (free-Hb, free-Hp or HpHb complex) was measured at a flow rate of 20 μl/min. Free-Hb and free-Hp were diluted to 1 μg/ml, 10 μg/ml and 100 μg/ml with HBS-EP running buffer (Cat. No. BR100188, GE Healthcare Bio-Sciences Corp.). HpHb complexes were prepared by incubating free-Hp and free-Hb at various concentrations, namely HpHb 10–50 (Hp 10 μg/ml and Hb 50 μg/ml), HpHb 50–50 (Hp 50 μg/ml and Hb 50 μg/ml), HpHb 100–50 (Hp 100 μg/ml and Hb 50 μg/ml), HpHb 50–10 (Hp 50 μg/ml and Hb 10 μg/ml), and HpHb 50–100 (Hp 50 μg/ml and Hb 100 μg/ml). rTcHpHbR and rTbHpHbR were diluted to 100 μg/ml with 10 mM sodium acetate, pH 4.5 (Cat. No. BR100350, GE Healthcare Bio-Sciences Corp.) and coupled to the surface of CM5 sensor chips (Cat. No. BR100530, GE healthcare Bio-Sciences Corp.). The final amounts of immobilized rTcHpHbR and rTbHpHbR were 5450 resonance units (RU) and 8000 RU, respectively.
The quantitative interaction between recombinant proteins and analytes was measured at a flow rate of 30 μl/min. Free-Hb and free-Hp were serially diluted to 10, 1, 0.1, 0.01, 0.001, 0.0001 μM as analyte solutions. rTcHpHbR and rTbHpHbR were diluted to 10 μg/ml with 10 mM sodium acetate (pH 4.5) and immobilized on the CM5 sensor chips. The final amounts of immobilized rTcHpHbR and rTbHpHbR were 800 RU and 480 RU respectively. All sensorgrams were approximated to the ideal curves by non-linear curve fitting, and the association rate constant and dissociation rate constant were calculated from the approximate curves using BIAevaluation software program (BIACORE Co., Ltd. Tokyo, Japan). The dissociation constant (K d) was calculated by dividing the dissociation rate constant by the association rate constant .
Cloning and the expression profile of TcHpHbR
Hemoglobin uptake in T. congolense
Direct interaction of TcHpHbR and free-hemoglobin
Binding parameters of TcHpHbR
Unlike other eukaryotes, trypanosomes obtain heme sources extracellularly as they lack a pathway for heme synthesis . As seen in Trypanosoma and Leishmania, heme uptake is important for the growth and development of the parasite [6, 22]. For example, TbHpHbR knockout mutants caused a decrease in the growth of T. brucei in mice . In contrast to the mammalian life-cycle stage, the vector life-cycle stages of trypanosomes require much higher amounts of heme as they need to produce heme proteins for an active electron transport chain . Thus hemoglobin uptake via hemoglobin receptors is essential for the survival of the vector life-cycle stages . Nevertheless, the mechanisms by which the vector life-cycle stages of African trypanosomes, such as the PCF and EMF, take up heme has not been well examined.
In the present study, we examined the expression profile, binding specificity and binding parameters of TcHpHbR, which was previously reported as a T. congolense orthologue of TbHpHbR [6, 8, 9]. Although TcHpHbR was described as a single copy gene, we showed that TcHpHbR was a 2 copy-gene with a tandem arrangement (Additional file 1: Figure S1 and Additional file 2: Figure S2). The amino acid sequences of TcHpHbR and TbHpHbR indicated that they shared 30 % identity (Fig. 1) . Higgins et al. reported that TcHpHbR was structurally similar to two well-characterized trypanosome GPI-anchored surface proteins (namely, VSG MITat 1.2 and GARP) in terms of their characteristic three-helical bundle . Nevertheless, TcHpHbR was exclusively transcribed and translated as 37 kDa and 42 kDa proteins in the EMF of T. congolense (Fig. 2). Presumably, the 37 kDa protein was unmodified TcHpHbR without the N-terminal signal peptide (Met1 to Val37), while the 42 kDa protein was post-translationally modified TcHpHbR. The molecular mass of TcHpHbR was similar to the predicted molecular mass of TbHpHbR (43.3 kDa), the apparent mass of which was 72 kDa because of N-glycosylation . Thus TcHpHbR appeared to have fewer post-translational modifications than TbHpHbR. This might be related to their different expression profiles during the parasite life-cycle. It was reported that TcHpHbR was structurally truncated in comparison to TbHpHbR , presumably because it does not need to protrude above a VSG layer, which is absent on the cell surface of EMF cells. Unlike TbHpHbR, the cellular localization of TcHpHbR was not limited to the flagellar pocket, rather it was found throughout the entire surface of EMF cells (Fig. 2c). However, since the uptake of Hb only occurred through the flagellar pocket (Fig. 3), cell surface TcHpHbR seemed to translocate to the flagellar pocket and to be endocytosed when it has bound Hb. This might suggest the presence of unique mechanisms that underlie the translocation of cell surface receptors to the flagella pocket in EMF. As we expected, the ligand specificity of TcHpHbR was also different from that of TbHpHbR. TbHpHbR is an HpHb complex-specific receptor , whereas TcHpHbR binds free-Hb with high affinity (Figs. 4 and 5a(4)). The ligand binding characteristics of TcHpHbR and TbHpHbR differed when the amount of free-Hp was exchanged for a specific amount of free-Hb (50 μg/ml) (Fig. 5b). According to this result, we speculated that the majority of free-Hb molecules could not form the HpHb complex in the tsetse midgut due to the lack of a sufficient amount of Hp molecules. Thus, the different ligand specificity of TcHpHbR and TbHpHbR may have evolved as a consequence of the adaptation of the different life-cycle stages of the two species to their different habitat within their vector. Since T. congolense EMFs occupy the proboscis of the tsetse fly vector, it seems that they are periodically exposed to a high level of free-Hb during blood meals. Hence, T. congolense EMFs may effectively take up free-Hb by the specific receptor, TcHpHbR. In contrast, T. brucei EMFs inhabit the salivary glands of their tsetse fly vector. Thus, the parasites do not come into contact with free-Hb.
We found that TcHpHbR, a TbHpHbR orthologue in T. congolense, was EMF-specific free-Hb receptor. We therefore propose that TcHpHbR should be renamed as T. congolense epimastigote-specific free-Hb receptor (TcEpHbR).
We thank Dr. Mo Zhou and Dr. Ngasaman Ruttayaporn for their technical assistance in the recombinant protein expression experiments. This work was supported by the Japan Society for the Promotion of Science (JSPS) (KAKENHI Grant Number 25292167).
SY and KS helped to conceive the study, participated in its design, and laboratory experiments. NY, SK and NI helped to conceive the study, participated in its design analyzed data, helped to draft and edit the manuscript and obtained funding. JY and MA helped to conceive the study, participated in its design, bioinformatics, and analyzed the data. All of the authors read and approved the final manuscript.
The authors declare that they have no competing interests.
The animal experiments were performed in accordance with the standards of animal experimentations in Obihiro University of Agriculture and Veterinary Medicine (No. 27–92).
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