TbVps41 regulates trafficking of endocytic but not biosynthetic cargo to lysosomes of bloodstream forms of Trypanosoma brucei

Abstract

The bloodstream stage of Trypanosoma brucei, the causative agent of African trypanosomiasis, is characterized by its high rate of endocytosis, which is involved in remodeling of its surface coat. Here we present evidence that RNAi-mediated expression down-regulation of vacuolar protein sorting 41 (Vps41), a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, leads to a strong inhibition of endocytosis, vesicle accumulation, enlargement of the flagellar pocket (“big eye” phenotype), and dramatic effect on cell growth. Unexpectedly, other functions described for Vps41 in mammalian cells and yeasts, such as delivery of proteins to lysosomes, and lysosome-related organelles (acidocalcisomes) were unaffected, indicating that in trypanosomes post-Golgi trafficking is distinct from that of mammalian cells and yeasts. The essentiality of TbVps41 suggests that it is a potential drug target.

1. INTRODUCTION

Trypanosoma brucei is a protist parasite and the causative agent of African trypanosomiasis or sleeping sickness. T. brucei bloodstream forms (BSF) thrive in the blood and extracellular fluids of mammals. The parasite is protected from the host immune response by periodically changing its surface coat of variant surface glycoprotein (VSG) by the mechanism of antigenic variation (1). VSG has a high rate of recycling involving endocytosis at the flagellar pocket, a mechanism that is also important to eliminate antibodies (2, 3). VSG coats are highly immunogenic and only parasites with a different VSG survive the host response. Blocking endocytosis by the BSF would be an efficient mechanism to eliminate the parasite without affecting the host. It has been pointed out that endocytosis could prevent antibody-dependent destruction of trypanosomes during the immune response, and then can be an important complementary mechanism to antigenic variation (2).

Studies in different eukaryotes have identified a number of proteins that are essential for endocytosis and traffic of cargo to the lysosomes (4). Among them, the class C core vacuole/endosome tethering factor (CORVET) that plays a role in early endosomes, and the late endosomal/lysosomal homotypic fusion and vacuole protein sorting (HOPS) complex, which is an endolysosomal tether essential for endolysosomal fusion, as well as for biosynthetic transport of proteins to lysosomes and lysosome related organelles. The CORVET/HOPS protein complexes have common core (Vps18, Vps11, Vps16, and Vps33) and specific subunits, Vps3/Vps8 for CORVET, and Vps39/Vps41 for HOPS (4). Interestingly, as occurs with other protists such as Toxoplasma gondii (5), neither Vps3 nor Vps8 homologues have been identified in the T. brucei genome, which suggest the absence of CORVET in these parasites. In addition to a role in endocytosis (4, 6, 7) and phagocytosis (8, 9), Vps41 plays an important role in adaptor protein complex 3 (AP-3)-mediated protein transport from the Golgi (10) or from endosomes (11, 12) to lysosomes and lysosome related organelles. Studies in yeast demonstrated that interaction of Vps41 with AP-3 complex δ subunit is crucial for AP-3 complex vesicle formation and intracellular protein trafficking (13). That study also demonstrated a direct interaction between Vps41 and the C-terminus of AP-3 δ subunit by yeast-two-hybrid analysis and co-immunoprecipitation. Further studies confirmed these results and determined that the N-terminal of Vps41 is necessary for this interaction (14, 15).

An endocytic route and a biosynthetic pathway to the lysosome has also been described in T. brucei (16), but only one subunit of HOPS, Vps41, was previously studied and only in the procyclic forms (PCF) found in the insect vector (17). That study proposed a role for TbVps41 in iron utilization and vacuolar fusion. Knockdown of its gene expression by RNAi did not affect growth, indicating that it is not essential in PCF (17).

T. brucei harbors lysosomes as well as lysosome related organelles termed the acidocalcisomes (18). Acidocalcisomes are acidic organelles that are rich in calcium and polyphosphate. Several proteins that localize to the acidocalcisomes are essential for parasite growth and survival (19–22). Acidocalcisome biogenesis in T. brucei (22) is mediated by the activity of AP-3 complex. We have previously shown that loss of AP-3 complex β3 or δ subunits resulted in loss of acidocalcisomes and immediate ablation of parasite growth in both insect (PCF) and mammalian (BSF) life cycle stages of the parasite (22). Owing to the connection between Vps41 and AP-3 complex and the key role of AP-3 complex in T. brucei acidocalcisome biogenesis, we evaluated the role of Vps41 in traffic of biosynthetic cargo to lysosomes and on acidocalcisome biogenesis.

In this work we found that RNAi silencing of TbVps41 resulted in a drastic defect in BSF growth. This effect was due to a strong inhibition of endocytosis with vesicle accumulation and enlargement of the flagellar pocket (“big eye” phenotype). However, in contrast to results in mammalian cells, other functions described for Vps41, such as delivery of proteins to lysosomes, and lysosome-related organelles (acidocalcisomes) were unaffected. The results provides further evidence that post-Golgi trafficking to the lysosome (16) and to the acidocalcisome (22) are distinct from these processes in other eukaryotic cells, suggesting lineage-specific adaptations during evolution.

2. MATERIALS AND METHODS:

2.1. Cell cultures

T. brucei BSF (wild-type (WT) and single marker (SM) strain, a gift from G.A.M Cross (Rockefeller University, N.Y.) (23) were cultivated at 37°C in HMI-9 medium (24) supplemented with 10% FBS and 2.5μg/mL of G418 (for SM).

2.2. Chemicals and Reagents

TRIzol reagent and SuperScript™ III First-Strand Synthesis System were purchased from Sigma. iQ SYBR green supermix was purchased from Bio-Rad. qPCR experiments were conducted on Bio-Rad CFX 96 real-time system, data was extracted using CFX manager 3.1 software from Bio-Rad and analyzed using Microsoft Excel and Graphpad Prism 5. The pMOTag4H vector (25) was a gift from Thomas Seebeck (University of Bern, Bern, Switzerland). The p2T7Ti vector was a gift from John Donelson (University of Iowa, Iowa City, IA). AMAXA Human T-cell Nucleofector kit was purchased from Lonza. The QIAgel extraction kit and MinElute PCR purification kit were from Qiagen. The primers were purchased from Integrated DNA Technologies. All other reagents of analytical grade were from Sigma.

2.3. Sequence analysis

Sequences for ScVps41 (NP_010365), HsVps41 (NP_055211.2), TbVps41 (XP_845398.1), ScAP-3δ (NP_015129.1) and TbAP-3δ (XP_845031.1) proteins were retrieved from NCBI database and analyzed using InterProScan analysis (https://www.ebi.ac.uk/interpro/search/sequence/). To characterize the motif/domains present in the Vps41 from Saccharomyces cerevisiae, Homo sapiens and T. brucei, we use InterproScan v.5.44 (26) with 15 different databases and also DELTA-BLAST. For visualization all Vps41 sequences with their respective annotated domains were aligned using MAFFT v.7.450 (27) to have their domains properly aligned for comparison. To construct the phylogenetic tree of AP-3, several orthologues from different euglenozoan species and two yeasts were retrieved from EuPathDB. These amino acid sequences were then aligned using MAFFT v.7.450 (27) and submitted to Modeltest-NG (28) to select the best substitution model for the Maximum Likelihood reconstruction. The model selected was Le-Gascuel with a discrete Gamma distribution with 5 rate categories (LG+G). The reconstruction was made using PhyML 3.3 (29) with 1000 bootstrap replicates. The Tree visualization was made using Figtree (Rambaut, 2009 - http://tree.bio.ed.ac.uk/software/figtree/).

2.4. Generation of TbVps41 RNAi constructs and C- or N-terminal epitope tagging cassettes

To knockdown the expression of the TbVps41 gene (XP_845398.1) by double-stranded RNA expression, the inducible T7 RNA polymerase-based protein expression system and the p2T7^Ti plasmid with dual-inducible T7 promoters were used. A 773-bp cDNA fragment of TbVps41 (nucleotides 2,267–3,040 of the ORF) was amplified using the primers indicated in Table S1 and cloned into the p2T7^Ti vector using HindIII and BamHI to generate p2T7(TbVps41). The recombinant construct p2T7(TbVps41) was confirmed by sequencing, NotI linearized, and then purified with QIAGEN’s DNA purification kit and used for cell transfections. The one-step epitope-tagging protocols reported by Oberholzer et al. (25) and Dean et al. (30) were used to produce C-terminal, smV5-tagging cassette of TbVps41 (XP_845398.1), and N-terminal, Ty-mNG-tagging cassettes of TbRab5A, TbRab7 and TbRab11 (XP_827782.1, XP_011776841.1, XP_847264.1, respectively) for transfection of T. brucei BSF WT 427 cell line. In brief, the PCR forward and reverse primers for C-terminal epitope tagging included terminal 100–120 nucleotides of each ORF before its stop codon and the reverse complement of the first 100–120 nucleotides of the 3’UTR, respectively, followed in frame by the 21–26 nucleotides of the backbone sequences of pMOTag2mV (31). The PCR forward and reverse primers for N-terminal epitope tagging included terminal 80 nucleotides of each 5’UTR before its start codon and the reverse complement of the first 80 nucleotides of the ORF after the start codon, respectively, followed in frame by the 18–20 nucleotides of the backbone sequences of pPOTv6-blast-blast-mNeonGreen (30), respectively. The smV5-or Ty-mNG-epitope tagging cassettes containing an antibiotic resistant gene as a selection marker (puromycin or blasticidin) were generated for cell transfection by PCR using pMOTag2mV (31) or pPOTv6-blast-blast-mNeonGreen (30), respectively, as template with the corresponding PCR primers of the gene (Table S1).

2.5. Cell transfection

Cell transfections were done as reported previously (20). In brief, 4 × 10⁷ mid-log phase BSF, 10 μg of cassette DNA or NotI-linearized plasmid DNA (in 10 μL) were used in 100 μL AMAXA Human T-cell Nucleofector solution. Electroporation was performed using 2 mm gap cuvettes with program X-001 of the AMAXA Nucleofector. Following each transfection, stable transformants were selected and cloned by limiting dilution in HMI-9 medium containing 15% FBS with 0.1 μg/mL puromycin, 5 μg/mL blasticidin or 2.5 μg/mL phleomycin in 24-well plates. The correct expression and regulation of smV5-epitope-tagged TbVps41, and Ty-mNG-tagged TbRab5A, TbRab7 or TbRab11 were confirmed by immunofluorescence and western blot analyses.

2.6. Antibodies

Guinea pig antibody against T. brucei vacuolar H⁺-pyrophosphatase (TbVP1) (32) was used at a concentration of 1:200 for immunofluorescence assays and 1:1000 for western blot analysis. Mouse anti-TbVTC4 antibody was used at 1:500 for western blot analysis (33). The rabbit polyclonal antibody against TbVP1 (1:300), mouse monoclonal antibody against V5 (1:100), rabbit polyclonal antibody against V5 (1:500), and mouse monoclonal antibody against Ty1 (1:200) were used for immunofluorescence assays. Alexa-conjugated secondary antibodies were purchased from Invitrogen. Tbp67 and TbCATL antibodies were a kind gift from Dr. James. D. Bangs (University of Buffalo, Buffalo, NY, USA) and were used at a concentration of 1:200 and 1:600 for immunofluorescence assays. Mouse monoclonal antibody against V5 (1:2500) and mouse monoclonal antibody against Ty1 (1:1000) were used for western blots. Rabbit and mouse anti-tubulin antibodies were used at 1:15000 for western blots. All secondary antibodies were used at 1:1000 for immunofluorescence assays and 1:15000 for western blots.

2.7. Western blot analysis

Mid-log phase T. brucei BSF were harvested by centrifugation at 1,000 x g for 10 min and washed twice with Buffer A with glucose (BAG, 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO₄, 50 mM HEPES, pH 7.2, and 5.5 mM glucose). The pellet was resuspended in 150 μl of RIPA buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% sodium dodecyl sulphate and 0.1% Triton X-100) supplemented with 2 mM phenylmethanesulfonyl fluoride (PMSF), and protease inhibitor cocktail (P8340, Sigma; 1:250 dilution). The lysates were incubated on ice for 1 h and then homogenized with a 1 ml syringe. Protein quantity was estimated using Pierce BCA protein assay kit. Thirty μg of protein from each lysate were separated by 10% SDS-PAGE using a Bio-Rad mini transblot cell. Precision plus protein western C was used as protein standard. Following electrophoresis, proteins were transferred to a nitrocellulose membrane using a Bio-Rad transblot apparatus at 100 V for 1 h. Membrane containing proteins was then blocked in 5% non-fat dry milk in PBST (PBS containing 0.1% v/v Tween-20) at RT for 1 h. Blots were then probed with primary antibody mouse anti-V5 from Thermo Fisher, or Guinea pig anti-TbVP1 (34) or Mouse anti-TbVTC4 (33) and rabbit anti-tubulin from Abcam or mouse anti-tubulin from Sigma at 4°C overnight. On the following day, the blots were washed with PBST three times and probed with secondary antibody IRDye goat anti-mouse 800CW or goat anti-rat 680 or goat anti-rabbit 680RD or donkey anti-guinea pig CF770 from LI-COR biosciences at RT for 1 h in dark. Following this, the membranes were washed again 3 times with PBST, imaged using Li-Cor Odyssey CLx imaging system and processed and analyzed using the image-studio software.

2.8. Immunofluorescence

To label flagellar pocket, live BSF were washed in ice-cold PBS with 1% glucose (PBS-G), and then incubated with 100 μg/ml of Concanavalin A (ConA)- tetramethylrhodamine conjugate (Thermo Fisher) in PBS-G on ice for 15 min. To determine the localization and expression of TbVps41 in T. brucei, BSF were washed in ice-cold PBS-G and then fixed with 1% paraformaldehyde in PBS at 4°C for 1 h. The fixed parasites were washed twice with PBS, allowed to adhere to poly-L-lysine-coated coverslips and permeabilized with 0.1% Triton X-100/PBS for 5 min. Following these the coverslips were washed again with PBS, pH 7.4 and blocked with PBST containing 5% goat serum, 50 mM NH₄Cl, 3% BSA, and 1% fish gelatin, at 4°C overnight. Next day, coverslips were incubated with primary antibody diluted in 1% BSA in PBS, pH 8.0 for 1 h, at RT, washed three times with 1% BSA in PBS, pH 8.0, and then incubated with secondary antibodies (1:1,000 dilution) diluted in 1% BSA in PBS, pH 8.0 for 1 h, at RT in the dark. Controls in the absence of primary antibodies were negative. The cells were washed three times with 1% BSA in PBS, pH 8.0, washed once with PBS, pH 8.0 and counterstained with DAPI (3 μg/ml) in Fluoromount-G mounting medium on the slides. Differential interference contrast (DIC) and fluorescent optical images (Figs. 1C and S1) were captured using an Olympus IX-71 inverted fluorescence microscope with a Photometrix CoolSnap CCD camera driven by DeltaVision software (Applied Precision). Images were deconvolved for 15 cycles using Softwarx deconvolution software. Fluorescent optical images (Figs. 3A–C and 5A–C) were taken with a 100 X oil immersion objective, a high-power solid-state 405 nm laser and EM-CCD camera (Andor iXon) (Andor Technology Ltd., Belfast, UK) under non-saturating conditions in a Zeiss ELYRA S1 (SR-SIM) super resolution microscope. Images were acquired and processed with ZEN 2011 software with SIM analysis module. The ELYRA S1 system achieves a lateral resolution of ~100 nm and an axial resolution of ~200 nm through the use of SR-SIM.

FIGURE 1.

Sequence analysis and localization of TbVps41. A, Schematic representation of various domains found in ScVps41, HsVps41 and TbVps41. Alignment of S. cerevisiae, H. sapiens, and T. brucei VPS41 protein sequences is shown. Motifs were detected with Interproscan using 15 different databases. The alignment was made using MAFFT v.7.450. Black lines represent sequenced aligned regions and thin lines are alignments gaps. B, Alignment made by MAFFT v.7.450 using S. cerevisiae and 10 different trypanosomatid Vps41 sequences from 6 different species. Showing conservation of the di-Leucine motif among all these species. The motif (D/E)XXXL(L/I) is not conserved in Leishmania sp. and Crithidia fasciculata, which possess a PXXXLL motif. C, Co-localization of TbVps41-smV5 (Vps41-smV5) with antibodies against Tbp67 (α-p67). Bars = 10 μm. Upper left image is a Differential Interference Contrast (DIC) microscopy image. Images are representative from 3 different experiments. D, Western blot analysis of smV5-tagged TbVps41. BSF trypanosomes were incubated with antibodies against V5 (α-V5), or tubulin (α-Tub), as a loading control, respectively. Markers are shown on the left, and the antibodies used in immunoblots are shown on the right

FIGURE 3.

Depletion of TbVps41 expression affects endocytosis in BSF trypanosomes. A-C, Cells were incubated with tomato lectin conjugated to biotin, washed, and then re-incubated to chase the lectin probe to the terminal endocytic compartments. Images were acquired from fixed and permeabilized cells stained with anti-p67 (red) and Alexa fluor 488:streptavidin (green). DAPI staining is in blue (merge). Bar = 10 μm. A, Fluorescence analysis of control cells grown in the absence of tetracycline. B, TbVps41-KD cells after 24 hours postinduction with tetracycline. C, TbVps41-KD cells after 48 hours postinduction with tetracycline. Images in A-C are representative of results from 3 independent experiments. D, Percentage of cells showing co-localization of anti-p67 and Alexa fluor 488:streptavidin in both control (-Tet) and TbVps41-KD parasites (+Tet) after 24 hours postinduction. At least 100 cells were counted in each experiment. Values are means ± s.d., n = 3, ****p < 0.0001. Statistical analyses were performed using one-way ANOVA and Turkey’s multiple comparisons test. E, Differential interference contrast (DIC) image of TbVps41-KD trypanosomes 48 hours postinduction showing enlargement of the flagellar pocket (big eye phenotype). Bar = 10 μm.

FIGURE 5.

Depletion of TbVps41 does not affect traffic of proteins to the acidocalcisomes or levels of acidocalcisome markers. A, Immunofluorescence analysis of control cells grown in the absence of tetracycline using antibodies against the acidocalcisome marker TbVP1 (red). DAPI staining is in blue in merge image. B, IFA of TbVps41-KD cells after 24 hours postinduction. C, IFA of TbVps41-KD cells after 48 hours postinduction. All images are representative of at least three independent experiments. Bars = 5 μm. D, Western blot analyses of BSF lysates of control and TbVps41-KD cells after 24 or 48 hours postinduction using anti-VP1 (α-VP1), anti-VTC4 (α-VTC4), and anti-tubulin antibodies (α-Tub) (loading control). All images are representative of those of at least three independent experiments.

2.9. Transmission electron microscopy

TbVps41 RNAi trypanosomes were either untreated or treated with 1 μg/ml tetracycline as indicated in the figure legends. Mid-log phase parasites were harvested by centrifugation at 1,000 x g for 10 min and fixed overnight at RT in 2.5% glutaraldehyde in 0.1 M cacodylate-HCl buffer, pH 7.2. Fixed parasites were rinsed in buffer and enrobed in 4% noble agar. Next, the parasites were post-fixed for 30 min with 1% OsO₄, 1.25% potassium ferrocyanide, and 5 mM CaCl₂ in 0.1 M cacodylate buffer, pH 7.2, dehydrated in an ascending ethanol series, and embedded in Epon resin. Ultrathin sections were generated and stained with uranyl acetate and lead citrate. Images were acquired at 80 kV using a JEOL JEM 1011 transmission electron microscope equipped with an XR80M Wide-Angle Multi-Discipline Mid-Mount CCD Camera from AMT (Advanced Microscopy Techniques) (AMT corp., Woburn, MA, USA).

2.10. Endocytosis assays

TbVps41 RNAi BSF were either untreated or treated with 1 μg/ml tetracycline for 24 hs as indicated in the figure legends. Endocytosis assays were performed as described previously (16). Briefly, the parasites were washed in HEPES-buffered saline (HBS; 50 mM HEPES-KOH, pH 7.5, 50 mM NaCl, 5 mM KCl, 70 mM glucose) and pre-incubated (10⁶ parasites/mL) in serum-free HMI-9 medium supplemented with BSA (0.5 mg/mL) at 37°C for 10 min followed by addition of tomato lectin conjugated to biotin (TL:biotin) for epifluorescence microscopy at a final concentrations of 5 μg/mL. Next, cells were incubated at 37°C for 30 min, washed, and incubated in fresh HMI-9 medium for 20 min to chase ligand into the terminal lysosome. In each case labeled cells were prepared, fixed and analyzed by immunofluorescence analysis. Endocytosis of tomato lectin conjugated to biotin was detected in fixed permeabilized cells by immunofluorescence assay using Strepatavidin:488.

2.11. Radioactive labelling and chase analyses

TbVps41 RNAi trypanosomes were either untreated or treated with 1 μg/ml tetracycline and used for pulse chase analysis. Pulse/chase analyses were performed using metabolic radiolabeling with [³⁵S]methionine/cysteine (Perkin Elmer) and subsequent immunoprecipitation of specific radiolabeled proteins from cell lysates as described previously (35, 36). For p67, pulse time was 15 min and for TbCATL pulse time was 10 min. Chase times are as indicated in the relevant figures. Immunoprecipitated proteins were analyzed by SDS-PAGE and phosphorimaging using a Typhoon FLA 9000 with native ImageQuant Software (GE Healthcare).

2.12. Statistical Analyses

All values are expressed as means + s.d. and “n” refers to the number of independent experiments performed. Statistical analyses were conducted using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA).

3. RESULTS

3.1. TbVps41 sequence analysis

The T. brucei genome contains a single TbVps41 gene (Tb427.06.2770, XP_845398.1) encoding a protein of 122 kDa. Analysis of the protein sequences for TbVps41 and for the S. cerevisiae (ScVps41) and human (HsVps41) orthologs was done using InterProScan (37). Two domains, WD40 repeat-like and CHCR domain, were found to be conserved in Vps41 from all three organisms (Fig. 1A). WD40 repeat-like domain is commonly found in proteins that form multiprotein complexes. The CHCR domain or clathrin heavy-chain repeat domain is commonly found in proteins that are involved in vacuolar maintenance, protein sorting and other clathrin-like functions. In addition to these domains, a PEST domain and an ALPS motif has been described for ScVps41 (38), but could not be detected using InterProScan. Of particular interest for our study was a PEST domain that contains the di-leucine motif, (D/E)XXXL(L/I), which is responsible for interaction with the C-terminus of AP3-δ subunit (13). A multiple sequence alignment of this region demonstrated the conservation of di-leucine motif in TbVps41 as well as Vps41 from several other trypanosomatids (Fig. 1B).

3.2. TbVps41 localization

In order to investigate the localization of TbVps41 in BSF, we tagged the endogenous copy of TbVps41 with a high-performance tag (spaghetti monster fluorescent protein [smFP] with V5 epitope tag) following the method of Oberholtzer et. al. (25) (Fig. 1C). Immunofluorescence microscopy analysis indicated dot-like staining for TbVps41 in BSF (Fig. 1C). Western blot analysis confirmed expression of TbVps41 of the expected size (Fig. 1D). TbVps41 did not localize to the lysosome as demonstrated by colocalization studies with lysosomal marker p67 (Fig. 1C). It did not colocalize to the flagellar pocket as shown by colocalization experiments with concanavalin (Fig. S1A). To determine if TbVps41 localizes to endosomes, we generated in situ Ty-mNG-tagged TbRab5A, TbRab7, and TbRab11 in smV5-tagged TbVps41 BSF cell line. Figs. S1B, S1C, and S1D show that TbVps41 did not localize to early, late, or recycling endosomes of BSF, as demonstrated by the lack of colocalization with Ty-mNG-tagged T. brucei endosomal marker proteins Rab5A, Rab7, and Rab11, respectively. Western blot analyses confirmed the expression of the Rab proteins with the expected apparent molecular weights (Figs. S1E, S1F and S1G).

3.3. TbVps41 RNAi

In order to determine the essentiality of TbVps41, we generated a cell line expressing a tetracycline inducible double stranded RNA (dsRNA) targeting the endogenous TbVps41. While the uninduced parasites grew normally over the course of 6 days, induction of TbVps41 silencing affected growth after 24 hours. Abnormal morphological changes and parasite death were observed until day 5. From day 5 on, despite induction of silencing, the parasites were able to recover and grow normally (Fig. 2A). Recovery from inducible dsRNA silencing (“escape”) has been reported previously for other genes in T. brucei BSF (39). Therefore, our results indicate that TbVps41 is required for normal growth of BSF. Downregulation of TbVps41 expression was confirmed by qRT-PCR analyses. At 24 hours about 65% reduction in TbVps41 mRNA could be observed, followed by a 90% reduction at 48 hours after induction (Fig. 2B).

FIGURE 2.

Effect of inhibition of TbVps41 expression by tetracycline-inducible RNAi on cell growth. A, Growth of BSF in the absence (blue filled circles) or presence (red filled squares) of 1 μg/mL tetracycline. Values are means ± s.d., n = 3. B, qRT-PCR analysis showing fold change of TbVps41-KD as compared to control in the absence of tetracycline taken at 24 and 48 h postinduction. Values are means ± s.d., n = 3, ****p < 0.0001. Statistical analyses were performed using one-way ANOVA and Turkey’s multiple comparisons test.

3.4. Endocytosis in TbVps41 knockdown mutants

To test if the TbVps41 silencing produces a defect in endocytosis, we used an endocytosis assay with biotinylated tomato lectin described previously (16). Biotinylated tomato lectin can serve as a marker for receptor-mediated endocytosis. In this assay, tomato lectin is endocytosed at the flagellar pocket and is transported to the lysosome. Co-localization of tomato lectin and the lysosome can be detected by immunostaining with streptavidin and p67 antibodies. When expression of TbVps41 was normal, tomato lectin exhibited excellent co-localization with p67 (Fig. 3A). However, after 24 hours of RNAi induction co-localization of internalized tomato lectin and p67 was markedly reduced (Fig. 3B), suggesting an early effect on endocytosis. Fig. 3D shows the quantification of co-localizations from multiple experiments. This effect was more accentuated after 48 hours of RNAi induction (Fig. 3C). However, in this case, the p67 labelling was distributed throughout the cell, indicative of vesicle accumulation, and the cell morphology was also affected (Fig. 3C). The appearance of multiple DAPI-stained kinetoplasts at 48 hours suggests that these cells have also failed to divide (Fig. 3C). After 48 hours of RNAi induction, enlarged flagellar pocket-like structures were also clearly visible (Fig. 3E). This phenotype appeared similar to the “big-eye” phenotype previously described in mutants of the endocytic pathway in T. brucei BSF (40, 41). Microscopy analyses of the TbVps41 knockdown (TbVps41-KD) cells at an earlier time point (24 hours) of RNAi induction found only 5 ± 1.5 % of the cells with the big eye phenotype (200 cells counted). Together these data strongly suggest that TbVps41 plays a role in the parasite endocytic pathway and that the loss of TbVps41 could affect parasite growth rate due to inefficient endocytosis by the parasites.

3.5. Flagellar pocket of TbVps41 RNAi in BSF

To further confirm the role of TbVps41 in T. brucei endocytosis, we subjected the parasites to electron microscopy (EM) analyses. As indicated above, the abnormal morphology associated with TbVps41 downregulation becomes more apparent after 36 hours of RNAi induction with about 50% of cells showing abnormal morphology (as in Fig. 3E). Therefore, we treated the parasites with tetracycline for 36 hours before subjecting them to EM analyses. Control parasites showed normal cellular structure with normal flagellar pockets (Fig. 4A). However, the TbVps41 depleted parasites had abnormally enlarged flagellar pockets and multiple vacuolation (Fig. 4B–C). Some of these vacuoles (Vac) contain electron-dense material and a double membrane (Fig. 4C, arrows), probably corresponding to autophagosomes.

FIGURE 4.

TbVps41 knockdown results in enlarged flagellar pocket. Images are representative of results from 3 independent experiments. A, Electron micrograph of control cells grown for 36 h in the absence of tetracycline. B, and C, Electron micrographs of TbVps41-KD trypanosomes for 36 h in the presence of tetracycline. F, flagellum; FP, flagellar pocket; K, kinetoplast; Vac., vacuole; arrows show vacuoles double membrane; N, nucleus. Bar = 0.6 μm.

3.6. Acidocalcisome biogenesis in TbVps41 RNAi is unaffected

Despite TbVps41 silencing, acidocalcisomes stained normally for the vacuolar proton pyrophosphatase (VP1), a marker of these organelles (Fig. 5A–C). Western blot analyses using antibodies for two acidocalcisome markers, VP1 and vacuolar transporter chaperone 4 (VTC4), revealed that TbVps41 RNAi did not appear to affect these proteins after 24 or 48 hours when compared to the tubulin control (Fig. 5D). As indicated before (Fig. 3C), the presence of multiple DAPI-stained kinetoplasts after 48 hours suggest that these cells failed to divide (Fig. 5C). Taken together these results suggest that the loss of TbVps41 does not affect acidocalcisome biogenesis.

3.7. Processing of proteins targeted to the lysosome is unaffected

Since TbVps41 appeared to play a role in endocytic trafficking to the lysosome, we further investigated if it was also involved in the transport of newly synthesized proteins to the lysosome. According to a previously described pulse/chase method (16), we monitored the processing of two proteins p67 and TbCATL targeted to the lysosomes. p67 in control cells is initially synthesized as a 100 kDa ER glycoform (gp100) and transported to the Golgi, N-glycan modified into a 150 kDa glycoform (gp150), followed by transport to lysosome where it is processed into quasi-stable 42 kDa (gp42) and 32 kDa (gp32) isoforms. We could observe a timely loss of gp100 isoform along with increase in quasi-stable gp42 and gp32 isoforms as the time progressed (Fig. 6A). However, the pattern was similar in TbVps41 depleted parasites as well. Quantification of band intensities also confirmed that the p67 trafficking was unaffected upon TbVps41 downregulation (Fig. 6B). We also assessed the processing of TbCATL. TbCATL is synthesized as a pre-pro-protein. The pre-pro-protein has a molecular weight of about 53 kDa (I) and is first processed into a pro-protein of about 50 kDa (X). The pro-protein is then processed into the mature protein, which has a molecular weight of about 40 kDa (M). As expected, in control cells we could observe the pre-pro-protein and pro-protein of TbCATL initially (Fig. 7A). Following the time-course, the bands corresponding to pre-pro-protein and pro-protein decrease in intensity while the band corresponding to the mature protein increases in intensity indicating proper trafficking of TbCATL to the lysosome of the control parasites (Fig. 7A, left lanes). When we compared this to the TbVps41 depleted parasites, we observed a similar trend to the control (Fig. 7A, right lanes). A statistical quantification of band intensities (Fig. 7B) further suggested that trafficking and processing of TbCATL was unaffected upon TbVps41 knockdown.

FIGURE 6.

Effect of TbVps41 silencing on Tbp67 trafficking to the lysosome. A, Transport of endogenous p67 was analyzed in control (-Tet) and TbVps41-KD (+Tet) trypanosomes grown for 24 hs. TbVps41-KD cells were pulse/chase radiolabeled with [³⁵S]Met/Cys. Then, samples were harvested at the indicated times and Tbp67 was immunoprecipitated from cell lysates and analyzed by SDS-PAGE/phosphoroimaging (10⁷ cell equivalents per lane). In each case p67 glycoforms can be observed as initial ER precursor (gp100); Golgi intermediate (gp150), lysosomal intermediate (gp75); quasi-stable lysosomal fragments (gp42 and gp32). B, The relative abundance of gp100, gp150, gp42, and gp32 as a percentage of the total immunoreactive proteins detected was quantified. Values are means ± s.d., n = 3. Statistical analyses were performed using Student’s t-test and was found to be non-significant.

FIGURE 7.

Effect of TbVps41 silencing on TbCATL traffic to the lysosome. A, Transport of endogenous TbCATL was analyzed in control (-Tet) and TbVps41-KD (+Tet) trypanosomes grown for 24 hs. TbVps41-KD cells were pulse/chase radiolabeled with [³⁵S]Met/Cys. Then, samples were harvested at the indicated times and TbCATL was immunoprecipitated from cell lysates and analyzed by SDS-PAGE/phosphoroimaging (10⁷ cell equivalents per lane). Arrival in the lysosome is indicated by loss of precursor (I + X) bands and appearance of mature (M) band. B, The relative amounts of I+X (blue circles and red squares) and M (blue triangles and red inverted triangles) bands were quantified. The data are presented as percentages of the original I+X and M bands and represent values are means ± s.d., n = 3. Statistical analyses were performed using Student’s t-test and was found to be non-significant.

4. DISCUSSION

Our work suggest that TbVps41 has a critical role in endocytic traffic to the lysosomes of T. brucei BSF without affecting post-Golgi traffic of proteins to lysosomes and acidocalcisomes. Knockdown of TbVps41 expression in BSF results in drastic effects on growth, revealing the essentiality of the endocytic traffic for the parasite survival.

Endocytosis in T. brucei BSF takes place at the flagellar pocket. Therefore, defects in endocytosis probably led to an enlarged flagellar pocket, or “big-eye” phenotype, also observed upon knockdown of expression of several T. brucei proteins involved in endocytosis, like the clathrin heavy chain, TbCLH, (40), actin (42), and the phosphatidylinositol 4,5-bisphosphate kinase, TbPIPKA, (41). Knockdown of TbVps41 for >24 h also resulted in vesicle accumulation. Results of endocytosis assays showed that knockdown of TbVps41 for 24 h prevented the delivery of endocytosed cargo to the lysosomes of the parasite, which were apparently fragmented, as shown by staining with p67 antibodies. That these were fragmented lysosomes, rather than late endosomes that failed to fuse with lysosomes, is suggested by the correct processing of p67 into 42 kDa (gp42) and 32 kDa (gp32) derivatives, which has been described to occur upon reaching the lysosomes (16). An alternative interpretation is that this processing is occurring in late endosomes that fail to interact with lysosomes. In this regard, Vps41 is part of the HOPS complex that, in yeast and animals, functions as tether between late endosomes and lysosomes and mediates their interaction. Vps41 is present on the outer interface of the HOPS complex and interacts with GTP bound Ypt7p (in yeast) or arl8b (in mammals) resulting in the tethering of late endosomes to lysosomes (9, 43–45). HOPS is a multiprotein complex comprising six proteins: Vps33, Vps16, Vps18, Vps11, Vps39 and Vps41 (46). Orthologs for all six proteins can be found in the T. brucei genome (Table S2). Interestingly, two orthologs for Vps39 can be found in the genomes of T. brucei, Trypanosoma cruzi and Leishmania major. It has been suggested that an extra copy of Vps39 substitutes the function of Vps3 in the CORVET complex in mammalian cells (47). Orthologs for Vps3 cannot be found in T. brucei, T. cruzi or L. major genomes. In this regard, HOPS/CORVET and related complexes exhibit quite significant evolutionary diversity in a broader context (48). The lack of Vps3 and Vps8 orthologues in T. brucei, which are the two CORVET Rab5-binding subunits (46) seems to indicate that the role of CORVET in the early endocytic pathway has been taken over by HOPS in trypanosomes.

Defects in HOPS complex components have been proposed to result in important pathologic conditions such as cancer, infectious diseases and neurological pathologies (8, 49–51). Despite such a universal significance of HOPS complex in all eukaryotes, its function in trypanosomatids has never been evaluated. Additionally, the interacting partner for Vps41 on the lysosome has not been clearly identified. In yeast, GTP bound Ypt7 has been demonstrated to interact with Vps41. However, in mammals a role for Ypt7 ortholog, Rab7, has not been demonstrated (45). Instead, other proteins such as arl8b or RILP have been proposed to interact with mammalian Vps41 to mediate lysosomal delivery of late endosomes (9, 45, 52, 53). Therefore, despite conservation of HOPS components across many eukaryotes, the binding partner for Vps41 might be unique in different organisms. Our results indicate a role for TbVps41 in parasite endocytic pathway and warrants the investigation of the HOPS complex in this trypanosomatid parasite.

Vps41, as part of the HOPS complex, has also been suggested to participate in trafficking of proteins to lysosomes and lysosomal-related organelles (54). Therefore, we tested the biosynthetic trafficking pathway of T. brucei to lysosomes by [³⁵S]Met/Cys pulse and chase analysis of two proteins, p67 and TbCATL (16, 22). Due to morphological and growth defect associated with TbVps41 RNAi mutants, we restricted the tetracycline treatment to 24 hours for these pulse chase assays. At this time point, TbVps41 mRNA was reduced by 70% but growth defects or morphological changes were not observed. Within this given time point we could not detect any defects in processing of p67 or TbCATL indicating that their trafficking and maturation were unaffected by TbVps41 knockdown.

Studies in yeast had identified that Vps41 plays a crucial role in AP-3 complex vesicle formation and function (13–15). AP-3 is involved in sorting of proteins to lysosomes and lysosomal-related organelles from the Golgi or from endosomes and is composed of two large subunits or adaptins (δ an β3), one medium adaptin (μ3), and one small subunit (σ3). ScVps41 contains a di-leucine motif within its PEST domain, which is responsible for interaction with AP-3 δ subunit. These PEST domain residues and the di-leucine motif appear to be conserved in TbVps41. The di-leucine motif in ScVps41 is responsible for interaction with the C-terminus (amino acids 711–932) of ScAP3-δ (14, 55). However, knockdown of TbVps41 did not affect trafficking of proteins to the lysosomes or acidocalcisomes. This can be attributed to the poor amino acid conservation of the C-terminal region of T. brucei AP-3 δ (Fig. S2). This suggests that TbVps41 may not be able to interact with TbAP-3 δ subunit. This could explain why the loss of TbAP-3 δ subunit affects acidocalcisome biogenesis (22) but the loss of TbVps41 has no such effect.

In conclusion, depletion of TbVps41 is lethal in BSF. In contrast, TbVps41 is non-essential in PCF (17) where the rate of endocytosis is much lower (56). Although in mammals Vps41 regulates AP-3-dependent traffic of proteins to the lysosome and lysosome-related organelles (54), this traffic is not affected by depletion of TbVps41. This is in agreement with results reporting that TbRab7 does not regulate the traffic of biosynthetic cargo to lysosomes of T. brucei (16). Rab7 (Ypt7) is the putative Vps41 receptor in yeast (45). The evidence presented demonstrates the distinct characteristics of post-Golgi traffic in trypanosomes, and suggests lineage specific adaptations. The essentiality of TbVps41, and the importance of endocytosis for T. brucei BSF suggest that it could be a potential drug target.

Abbreviations:

AP-3

adaptor protein complex 3

BSA

bovine serum albumin

BSF

bloodstream forms

CORVET

class C core vacuole/endosome tethering factor

PCF

procyclic forms

HOPS

homotypic fusion and vacuole protein sorting

PBS

phosphate buffer saline

VAC

vacuole

VP1

vacuolar proton pyrophosphatase

Vps41

vacuolar protein sorting 41

VSG

variant surface glycoprotein