Dynamic, variable oligomerization and the trafficking of variant surface glycoproteins of Trypanosoma brucei.

African trypanosomes cause disease in humans and livestock, avoiding host immunity by changing the expression of variant surface glycoproteins (VSGs); the major glycosylphosphatidylinositol (GPI) anchored antigens coating the surface of the bloodstream stage. Proper trafficking of VSGs is therefore critical to pathogen survival. The valence model argues that GPI anchors regulate progression and fate in the secretory pathway and that, specifically, a valence of two (VSGs are dimers) is critical for stable cell surface association. However, recent reports that the MITat1.3 (M1.3) VSG N-terminal domain (NTD) behaves as a monomer in solution and in a crystal structure challenge this model. We now show that the behavior of intact M1.3 VSG in standard in vivo trafficking assays is consistent with an oligomer. Nevertheless, Blue Native Gel electrophoresis and size exclusion chromatography-multiangle light scattering chromatography of purified full length M1.3 VSG indicates a monomer in vitro. However, studies with additional VSGs show that multiple oligomeric states are possible, and that for some VSGs oligomerization is concentration dependent. These data argue that individual VSG monomers possess different propensities to self-oligomerize, but that when constrained at high density to the cell surface, oligomeric species predominate. These results resolve the apparent conflict between the valence hypothesis and the M1.3 NTD VSG crystal structure.

Steric constraints control processing of glycosylphosphatidylinositol anchors in Trypanosoma brucei.

The transferrin receptor (TfR) of the bloodstream form (BSF) of Trypanosoma brucei is a heterodimer comprising glycosylphosphatidylinositol (GPI)-anchored expression site-associated gene 6 (ESAG6 or E6) and soluble ESAG7. Mature E6 has five N-glycans, consisting of three oligomannose and two unprocessed paucimannose structures. Its GPI anchor is modified by the addition of 4-6 α-galactose residues. TfR binds tomato lectin (TL), specific for N-acetyllactosamine (LacNAc) repeats, and previous studies have shown transport-dependent increases in E6 size consistent with post-glycan processing in the endoplasmic reticulum. Using pulse-chase radiolabeling, peptide-N-glycosidase F treatment, lectin pulldowns, and exoglycosidase treatment, we have now investigated TfR N-glycan and GPI processing. E6 increased ∼5 kDa during maturation, becoming reactive with both TL and Erythrina cristagalli lectin (ECL, terminal LacNAc), indicating synthesis of poly-LacNAc on paucimannose N-glycans. This processing was lost after exoglycosidase treatment and after RNAi-based silencing of TbSTT3A, the oligosaccharyltransferase that transfers paucimannose structures to nascent secretory polypeptides. These results contradict previous structural studies. Minor GPI processing was also observed, consistent with α-galactose addition. However, increasing the spacing between E6 protein and the GPI ω-site (aa 4-7) resulted in extensive post-translational processing of the GPI anchor to a form that was TL/ECL-reactive, suggesting the addition of LacNAc structures, confirmed by identical assays with BiPNHP, a non-N-glycosylated GPI-anchored reporter. We conclude that BSF trypanosomes can modify GPIs by generating structures reminiscent of those present in insect-stage trypanosomes and that steric constraints, not stage-specific expression of glycosyltransferases, regulate GPI processing.

Late ESCRT machinery mediates the recycling and Rescue of Invariant Surface Glycoprotein 65 in Trypanosoma brucei.

The Endosomal Sorting Complex Required for Transport machinery consists of four protein complexes (ESCRT 0-IV) and the post ESCRT ATPase Vps4. ESCRT mediates cargo delivery for lysosomal degradation via formation of multivesicular bodies. Trypanosoma brucei contains orthologues of ESCRT I-III and Vps4. Trypanosomes also have an ubiquitinylated invariant surface glycoprotein (ISG65) that is delivered to the lysosome by ESCRT, however, we previously implicated TbVps4 in rescue and recycling of ISG65. Here we use conditional silencing to investigate the role of TbVps24, a phosphoinositide-binding ESCRT III component, on protein trafficking. TbVps24 localises to the TbRab7+ late endosome, and binds PI(3,5)P2 , the product of the TbFab1 kinase, both of which also localise to late endosomes. TbVps24 silencing is lethal, and negatively affects biosynthetic trafficking of the lysosomal markers p67 and TbCathepsin L. However, the major phenotype of silencing is accelerated degradation and depletion of the surface pool of ISG65. Thus, TbVps24 silencing phenocopies that of TbVps4 in regard to ISG65 trafficking. This presents a paradox since we have previously found that depletion of TbFab1 completely blocks ISG65 turnover. We propose a model in which late ESCRT components operate at two sites, one PI(3,5)P2 -dependent (degradation) and one PI(3,5)P2 -independent (recycling), to regulate ISG65 homeostasis.

p67: a cryptic lysosomal hydrolase in Trypanosoma brucei?

p67 is a type I transmembrane glycoprotein of the terminal lysosome of African trypanosomes. Its biosynthesis involves transport of an initial gp100 ER precursor to the lysosome, followed by cleavage to N-terminal (gp32) and C-terminal (gp42) subunits that remain non-covalently associated. p67 knockdown is lethal, but the only overt phenotype is an enlarged lysosome (~250 to >1000 nm). Orthologues have been characterized in Dictyostelium and mammals. These have processing pathways similar to p67, and are thought to have phospholipase B-like (PLBL) activity. The mouse PLBD2 crystal structure revealed that the PLBLs represent a subgroup of the larger N-terminal nucleophile (NTN) superfamily, all of which are hydrolases. NTNs activate by internal autocleavage mediated by a nucleophilic residue, i.e. Cys, Ser or Thr, on the upstream peptide bond to form N-terminal α (gp32) and C-terminal ß (gp42) subunits that remain non-covalently associated. The N-terminal residue of the ß subunit is then catalytic in subsequent hydrolysis reactions. All PLBLs have a conserved Cys/Ser dipeptide at the α/ß junction (Cys241/Ser242 in p67), mutation of which renders p67 non-functional in RNAi rescue assays. p67 orthologues are found in many clades of parasitic protozoa, thus p67 is the founding member of a group of hydrolases that likely play a role broadly in the pathogenesis of parasitic infections.

Rab11 mediates selective recycling and endocytic trafficking in Trypanosoma brucei.

Trypanosoma brucei possesses a streamlined secretory system that guarantees efficient delivery to the cell surface of the critical glycosyl-phosphatidylinositol (GPI)-anchored virulence factors, variant surface glycoprotein (VSG) and transferrin receptor (TfR). Both are thought to be constitutively endocytosed and returned to the flagellar pocket via TbRab11+ recycling endosomes. We use conditional knockdown with established reporters to investigate the role of TbRab11 in specific endomembrane trafficking pathways in bloodstream trypanosomes. TbRab11 is essential. Ablation has a modest negative effect on general endocytosis, but does not affect turnover, steady state levels or surface localization of TfR. Nor are biosynthetic delivery to the cell surface and recycling of VSG affected. TbRab11 depletion also causes increased shedding of VSG into the media by formation of nanotubes and extracellular vesicles. In contrast to GPI-anchored cargo, TbRab11 depletion reduces recycling of the transmembrane invariant surface protein, ISG65, leading to increased lysosomal turnover. Thus, TbRab11 plays a critical role in recycling of transmembrane, but not GPI-anchored surface proteins. We proposed a two-step model for VSG turnover involving release of VSG-containing vesicles followed by GPI hydrolysis. Collectively, our results indicate a critical role of TbRab11 in the homeostatic maintenance of the secretory/endocytic system of bloodstream T. brucei.

Processing and targeting of cathepsin L (TbCatL) to the lysosome in Trypanosoma brucei.

Cathepsin L (TbCatL) is an essential lysosomal thiol protease in African trypanosomes. TbCatL is synthesized as two precursor forms (P/X) that are activated to mature form (M) with the removal of the prodomain upon arrival in the lysosome. We examine TbCatL trafficking in a novel system: truncated TbCatL reporter without the C-terminal domain (CTD; TbCatL∆) ectopically expressed in an RNA interference (RNAi) cell line targeting the CTD/3' untranslated region (UTR) of endogenous mRNA. TbCatL∆ is synthesized as P'/X'/M' species, localizes to the lysosome, and rescues the lethal TbCatL RNAi phenotype. Inactive TbCatLΔ:C150A is only processed to M' in the presence of endogenous TbCatL indicating trans-auto-catalytic activation. X' is formed with active endoplasmic reticulum (ER)-retained TbCatLΔ:MDDL, but not with TbCatLΔ:C150A, indicating stochastic generation in the ER by cis-auto-cleavage within the prodomain of newly synthesized P'. Modelling the TbCatL prodomain on the human CatL structure suggests three solvent accessible features that could contain post-Golgi targeting signals: the N-terminus, the helix 1/turn 1 junction, and a separate turn (T3). We demonstrate that the critical motif for lysosomal targeting is an asparagine-proline dipeptide in T3 that is strictly conserved in all Kinetoplastida. These findings show novel insights on the maturation of TbCatL, which is a critical virulence factor in mammalian infection.

Evolution of Antigenic Variation in African Trypanosomes: Variant Surface Glycoprotein Expression, Structure, and Function.

The process of antigenic variation in parasitic African trypanosomes is a remarkable mechanism for outwitting the immune system of the mammalian host, but it requires a delicate balancing act for the monoallelic expression, folding and transport of a single variant surface glycoprotein (VSG). Only one of hundreds of VSG genes is expressed at time, and this from just one of ≈15 dedicated expression sites. By switching expression of VSGs the parasite presents a continuously shifting antigenic facade leading to prolonged chronic infections lasting months to years. The basics of VSG structure and switching have been known for several decades, but recent studies have brought higher resolution to many aspects this process. New VSG structures, in silico modeling of infections, studies of VSG codon usage, and experimental ablation of VSG expression provide insights that inform how this remarkable system may have evolved.

Controlling transferrin receptor trafficking with GPI-valence in bloodstream stage African trypanosomes.

Bloodstream-form African trypanosomes encode two structurally related glycosylphosphatidylinositol (GPI)-anchored proteins that are critical virulence factors, variant surface glycoprotein (VSG) for antigenic variation and transferrin receptor (TfR) for iron acquisition. Both are transcribed from the active telomeric expression site. VSG is a GPI2 homodimer; TfR is a GPI1 heterodimer of GPI-anchored ESAG6 and ESAG7. GPI-valence correlates with secretory progression and fate in bloodstream trypanosomes: VSG (GPI2) is a surface protein; truncated VSG (GPI0) is degraded in the lysosome; and native TfR (GPI1) localizes in the flagellar pocket. Tf:Fe starvation results in up-regulation and redistribution of TfR to the plasma membrane suggesting a saturable mechanism for flagellar pocket retention. However, because such surface TfR is non-functional for ligand binding we proposed that it represents GPI2 ESAG6 homodimers that are unable to bind transferrin-thereby mimicking native VSG. We now exploit a novel RNAi system for simultaneous lethal silencing of all native TfR subunits and exclusive in-situ expression of RNAi-resistant TfR variants with valences of GPI0-2. Our results conform to the valence model: GPI0 ESAG7 homodimers traffick to the lysosome and GPI2 ESAG6 homodimers to the cell surface. However, when expressed alone ESAG6 is up-regulated ~7-fold, leaving the issue of saturable retention in the flagellar pocket in question. Therefore, we created an RNAi-resistant GPI2 TfR heterodimer by fusing the C-terminal domain of ESAG6 to ESAG7. Co-expression with ESAG6 generates a functional heterodimeric GPI2 TfR that restores Tf uptake and cell viability, and localizes to the cell surface, without overexpression. These results resolve the longstanding issue of TfR trafficking under over-expression and confirm GPI valence as a critical determinant of intracellular sorting in trypanosomes.

Mammalian African trypanosome VSG coat enhances tsetse's vector competence.

Tsetse flies are biological vectors of African trypanosomes, the protozoan parasites responsible for causing human and animal trypanosomiases across sub-Saharan Africa. Currently, no vaccines are available for disease prevention due to antigenic variation of the Variant Surface Glycoproteins (VSG) that coat parasites while they reside within mammalian hosts. As a result, interference with parasite development in the tsetse vector is being explored to reduce disease transmission. A major bottleneck to infection occurs as parasites attempt to colonize tsetse's midgut. One critical factor influencing this bottleneck is the fly's peritrophic matrix (PM), a semipermeable, chitinous barrier that lines the midgut. The mechanisms that enable trypanosomes to cross this barrier are currently unknown. Here, we determined that as parasites enter the tsetse's gut, VSG molecules released from trypanosomes are internalized by cells of the cardia-the tissue responsible for producing the PM. VSG internalization results in decreased expression of a tsetse microRNA (mir-275) and interferes with the Wnt-signaling pathway and the Iroquois/IRX transcription factor family. This interference reduces the function of the PM barrier and promotes parasite colonization of the gut early in the infection process. Manipulation of the insect midgut homeostasis by the mammalian parasite coat proteins is a novel function and indicates that VSG serves a dual role in trypanosome biology-that of facilitating transmission through its mammalian host and insect vector. We detail critical steps in the course of trypanosome infection establishment that can serve as novel targets to reduce the tsetse's vector competence and disease transmission.

Surface proteins, ERAD and antigenic variation in Trypanosoma brucei.

Variant surface glycoprotein (VSG) is central to antigenic variation in African trypanosomes. Although much prior work documents that VSG is efficiently synthesized and exported to the cell surface, it was recently claimed that 2-3 fold more is synthesized than required, the excess being eliminated by ER-Associated Degradation (ERAD) (Field et al., ). We now reinvestigate VSG turnover and find no evidence for rapid degradation, consistent with a model whereby VSG synthesis is precisely regulated to match requirements for a functional surface coat on each daughter cell. However, using a mutated version of the ESAG7 subunit of the transferrin receptor (E7:Ty) we confirm functional ERAD in trypanosomes. E7:Ty fails to assemble into transferrin receptors and accumulates in the ER, consistent with retention of misfolded protein, and its turnover is selectively rescued by the proteasomal inhibitor MG132. We also show that ER accumulation of E7:Ty does not induce an unfolded protein response. These data, along with the presence of ERAD orthologues in the Trypanosoma brucei genome, confirm ERAD in trypanosomes. We discuss scenarios in which ERAD could be critical to bloodstream parasites, and how these may have contributed to the evolution of antigenic variation in trypanosomes.

Unfolded Protein Response Pathways in Bloodstream-Form Trypanosoma brucei?

The unfolded protein response (UPR) is a stress mechanism to cope with misfolded proteins in the early secretory pathway, the hallmark being transcriptional upregulation of endoplasmic reticulum (ER) molecular chaperones such as BiP and protein disulfide isomerase. Despite the lack of transcriptional regulation and the absence of the classical UPR machinery, African trypanosomes apparently respond to persistent ER stress by a UPR-like response, including upregulation of BiP, and a related spliced leader silencing (SLS) response whereby SL RNA transcription is shut down. Initially observed by knockdown of the secretory protein translocation machinery, both responses are also induced by chemical agents known to elicit UPR in mammalian cells (H. Goldshmidt, D. Matas, A. Kabi, A. Carmi, R. Hope, S. Michaeli, PLoS Pathog 6:e1000731, 2010, http://dx.doi.org/10.1371/journal.ppat.1000731). As these findings were generated primarily in procyclic-stage trypanosomes, we have investigated both responses in pathogenic bloodstream-stage parasites. RNA interference (RNAi) silencing of the core translocon subunit Trypanosoma brucei Sec61α (TbSec61α) failed to induce either response. Interestingly, cell growth halted within 16 h of silencing, but sufficient TbSec61α remained to allow full competence for translocation of nascent secretory proteins for up to 24 h, indicating that replication is finely coupled with the capacity to synthesize and transport secretory cargo. Tunicamycin and thapsigargin at concentrations compatible with short-term (4 h) and long-term (24 h) viability also failed to induce any of the indicators of UPR-like or SLS responses. Dithiothreitol (DTT) was lethal at all concentrations tested. These results indicate that UPR-like and SLS responses to persistent ER stress do not occur in bloodstream-stage trypanosomes.

Characterization of the late endosomal ESCRT machinery in Trypanosoma brucei.

The multivesicular body (MVB) is a specialized Rab7+ late endosome (LE) containing multiple intralumenal vesicles that function in targeting ubiquitinylated cell surface proteins to the lysosome for degradation. African trypanosomes lack a morphologically well-defined MVB, but contain orthologs of the ESCRT (Endosomal Sorting Complex Required for Transport) machinery that mediates MVB formation. We investigate the role of TbVps23, an early ESCRT component, and TbVps4, the terminal ESCRT ATPase, in lysosomal trafficking in bloodstream form trypanosomes. Both localize to the TbRab7+ LE and RNAi silencing of each rapidly blocks growth. TbVps4 silencing results in approximately threefold accumulation of TbVps23 at the LE, consistent with blocking terminal ESCRT disassembly. Trafficking of endocytic and biosynthetic cargo, but not default lysosomal reporters, is also negatively affected. Others reported that TbVps23 mediates ubiquitin-dependent lysosomal degradation of invariant surface glycoproteins (ISG65) (Leung et al., Traffic 2008;9:1698-1716). In contrast, we find that TbVps23 ablation does not affect ISG65 turnover, while TbVps4 silencing markedly enhances lysosomal degradation. We propose several models to accommodate these results, including that the ESCRT machinery actually retrieves ISG65 from the LE to earlier endocytic compartments, and in its absence ISG65 traffics more efficiently to the lysosome. Overall, these results confirm that the ESCRT machinery is essential in Trypanosoma brucei and plays important and novel role(s) in LE function in trypanosomes.

Inhibition of nucleotide sugar transport in Trypanosoma brucei alters surface glycosylation.

Nucleotide sugar transporters (NSTs) are indispensible for the biosynthesis of glycoproteins by providing the nucleotide sugars needed for glycosylation in the lumen of the Golgi apparatus. Mutations in NST genes cause human and cattle diseases and impaired cell walls of yeast and fungi. Information regarding their function in the protozoan parasite, Trypanosoma brucei, a causative agent of African trypanosomiasis, is unknown. Here, we characterized the substrate specificities of four NSTs, TbNST1-4, which are expressed in both the insect procyclic form (PCF) and mammalian bloodstream form (BSF) stages. TbNST1/2 transports UDP-Gal/UDP-GlcNAc, TbNST3 transports GDP-Man, and TbNST4 transports UDP-GlcNAc, UDP-GalNAc, and GDP-Man. TbNST4 is the first NST shown to transport both pyrimidine and purine nucleotide sugars and is demonstrated here to be localized at the Golgi apparatus. RNAi-mediated silencing of TbNST4 in the procyclic form caused underglycosylated surface glycoprotein EP-procyclin. Similarly, defective glycosylation of the variant surface glycoprotein (VSG221) as well as the lysosomal membrane protein p67 was observed in Δtbnst4 BSF T. brucei. Relative infectivity analysis showed that defects in glycosylation of the surface coat resulting from tbnst4 deletion were insufficient to impact the ability of this parasite to infect mice. Notably, the fact that inactivation of a single NST gene results in measurable defects in surface glycoproteins in different life cycle stages of the parasite highlights the essential role of NST(s) in glycosylation of T. brucei. Thus, results presented in this study provide a framework for conducting functional analyses of other NSTs identified in T. brucei.

Glycoprotein biosynthesis in a eukaryote lacking the membrane protein Rft1.

Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 is intimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLO flippase, but because biochemical tests challenged this assignment, the function of Rft1 remains obscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis in vivo in the complete absence of the protein. Rft1 is essential for yeast viability, and no Rft1-null organisms are currently available. Here, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report that TbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels of mDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity. Remarkably, the mutant cells have 30-100-fold greater steady-state levels of M5-DLO than wild-type cells. All N-glycans in the TbRft1-null cells originate from mDLO indicating that the M5-DLO excess is not available for glycosylation. These results suggest that rather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by another mechanism, possibly by acting as an M5-DLO chaperone.

Intracellular trafficking and glycobiology of TbPDI2, a stage-specific protein disulfide isomerase in Trypanosoma brucei.

Trypanosoma brucei protein disulfide isomerase 2 (TbPDI2) is a bloodstream stage-specific lumenal endoplasmic reticulum (ER) glycoprotein. ER localization is dependent on the TbPDI2 C-terminal tetrapeptide (KQDL) and is mediated by TbERD2, an orthologue of the yeast ER retrieval receptor. Consistent with this function, TbERD2 localizes prominently to ER exit sites, and RNA interference (RNAi) knockdown results in specific secretion of a surrogate ER retention reporter, BiPN:KQDL. TbPDI2 is highly N-glycosylated and is reactive with tomato lectin, suggesting the presence of poly-N-acetyllactosamine modifications, which are common on lyso/endosomal proteins in trypanosomes but are inconsistent with ER localization. However, TbPDI2 is reactive with tomato lectin immediately following biosynthesis-far too rapidly for transport to the Golgi compartment, the site of poly-N-acetyllactosamine addition. TbPDI2 also fails to react with Erythrina cristagalli lectin, confirming the absence of terminal N-acetyllactosamine units. We propose that tomato lectin binds the Manß1-4GlcNAcß1-4GlcNAc trisaccharide core of paucimannose glycans on both newly synthesized and mature TbPDI2. Consistent with this proposal, α-mannosidase treatment renders oligomannose N-glycans on the T. brucei cathepsin L orthologue TbCatL reactive with tomato lectin. These findings resolve contradictory evidence on the location and glycobiology of TbPDI2 and provide a cautionary note on the use of tomato lectin as a poly-N-acetyllactosamine-specific reagent.

Replication of the ERES:Golgi junction in bloodstream-form African trypanosomes.

The biogenesis of the ER Exit Site/Golgi Junction (EGJ) in bloodstream-form African trypanosomes is investigated using tagged markers for ER Exit Sites, the Golgi and the bilobe structure. The typical pattern is two EGJ in G1 phase (1 kinetoplast/1 nucleus, 1K1N) through S-phase (2K1N), duplication to four EGJ in post-mitotic cells (2K2N) and segregation of two EGJ to each daughter. Lesser cell percentages have elevated EGJ copy numbers in all stages, and blocking cell cycle progression results in even higher copy numbers. EGJs are closely aligned with the flagellar attachment zone (FAZ) indicating nucleation on the FAZ-associated ER (FAZ:ER). Only the most posterior EGJ in each cell is in proximity to the bilobe, which is located at the base of the FAZ filament near the mouth of the flagellar pocket. These results indicate that EGJ replication in bloodstream trypanosomes is not tightly coupled to the cell cycle. Furthermore, segregation of EGJ is not obligately mediated by the bilobe, rather assembly of the EGJ on the FAZ:ER, which is coupled to the flagellar cytoskeleton, apparently ensures segregation with fidelity during cytokinesis. These findings differ markedly from procyclic-form trypanosomes, and models highlighting these stage-specific differences in EGJ biogenesis are proposed.

Late endosomal Rab7 regulates lysosomal trafficking of endocytic but not biosynthetic cargo in Trypanosoma brucei.

We present the first functional analysis of the small GTPase, TbRab7, in Trypanosoma brucei. TbRab7 defines discrete late endosomes closely juxtaposed to the terminal p67(+) lysosome. RNAi indicates that TbRab7 is essential in bloodstream trypanosomes. Initial rates of endocytosis were unaffected, but lysosomal delivery of cargo, including tomato lectin (TL) and trypanolytic factor (TLF) were blocked. These accumulate in a dispersed internal compartment of elevated pH, likely derived from the late endosome. Surface binding of TL but not TLF was reduced, suggesting that cellular distribution of flagellar pocket receptors is differentially regulated by TbRab7. TLF activity was reduced approximately threefold confirming that lysosomal delivery is critical for trypanotoxicity. Unexpectedly, delivery of endogenous proteins, p67 and TbCatL, were unaffected indicating that TbRab7 does not regulate biosynthetic lysosomal trafficking. Thus, unlike mammalian cells and yeast, lysosomal trafficking of endocytosed and endogenous proteins occur via different routes and/or are regulated differentially. TbRab7 silencing had no effect on a cryptic default pathway to the lysosome, suggesting that the default lysosomal reporters p67ΔTM, p67ΔCD and VSGΔGPI do not utilize the endocytic pathway as previously proposed. Surprisingly, conditional knockout indicates that TbRab7 may be non-essential in procyclic insect form trypanosomes.

Stage-Specific COPII-Mediated Cargo Selectivity in African Trypanosomes.

A hallmark of eukaryotic cells is the ability to form a secretory pathway connecting many intracellular compartments. In the early secretory pathway, coated protein complex II (COPII)-coated vesicles mediate the anterograde transport of newly synthesized secretory cargo from the endoplasmic reticulum to the Golgi apparatus. The COPII coat complex is comprised of an inner layer of Sec23/Sec24 heterodimers and an outer layer of Sec13/Sec31 heterotetramers. In African trypanosomes, there are two paralogues each of Sec23 and Sec24, that form obligate heterodimers (TbSec23.2/TbSec24.1, TbSec23.1/TbSec24.2). It is not known if these form distinct homotypic classes of vesicles or one heterotypic class, but it is known that TbSec23.2/TbSec24.1 specifically mediate forward trafficking of GPI-anchored proteins (GPI-APs) in bloodstream-form trypanosomes (BSF). Here, we showed that this selectivity was lost in insect procyclic stage parasites (PCF). All isoforms of TbSec23 and TbSec24 are essential in PCF parasites as judged by RNAi knockdowns. RNAi silencing of each subunit had equivalent effects on the trafficking of GPI-APs and p67, a transmembrane lysosomal protein. However, silencing of the TbSec23.2/TbSec24.1 had heterodimer had a significant impact on COPII mediated trafficking of soluble TbCatL from the ER to the lysosome. This finding suggests a model in which selectivity of COPII transport was altered between the BSF and PCF trypanosomes, possibly as an adaptation to a digenetic life cycle. IMPORTANCE African trypanosomes synthesize dense surface coats composed of stage-specific glycosylphosphatidylinositol lipid anchored proteins. We previously defined specific machinery in bloodstream stage parasites that mediate the exit of these proteins from the endoplasmic reticulum. Here, we performed similar analyses in the procyclic insect stage and found significant differences in this process. These findings contribute to our understanding of secretory processes in this unusual eukaryotic model system.

Turnover of Variant Surface Glycoprotein in Trypanosoma brucei Is Not Altered in Response to Specific Silencing.

African trypanosomes evade the immune system of the mammalian host by the antigenic variation of the predominant glycosylphosphatidylinositol (GPI)-anchored surface protein, variant surface glycoprotein (VSG). VSG is a very stable protein that is turned over from the cell surface with a long half-life (~26 h), allowing newly synthesized VSG to populate the surface. We have recently demonstrated that VSG turnover under normal growth is mediated by a combination of GPI hydrolysis and direct shedding with intact GPI anchors. VSG synthesis is tightly regulated in dividing trypanosomes, and when subjected to RNA interference (RNAi) silencing, cells display rapid cell cycle arrest in order to conserve VSG density on the cell surface (K. Sheader, S. Vaughan, J. Minchin, K. Hughes, et al., Proc Natl Acad Sci U S A 102:8716-8721, 2005, https://doi.org/10.1073/pnas.0501886102). Arrested cells also display an altered morphology of secretory organelles-engorgement of the trans-Golgi cisternae-that may reflect a disruption of post-Golgi secretory transport. We now ask whether trypanosomes under VSG silencing also reduce the rate of VSG turnover to further conserve coat density. Our data indicate that trypanosomes do not regulate VSG turnover according to VSG protein abundance, nor was there any effect on the post-Golgi transport of soluble or GPI-anchored secretory cargo. However, the surface morphology of silenced cells was altered from a typically rugose topology to a smoother profile, consistent with reduced overall membrane trafficking to the cell surface. IMPORTANCE African trypanosomes evade the host immune system by altering the expression of variant surface glycoproteins (VSGs) in a process called antigenic variation. VSG is essential, and when its synthesis is ablated by RNAi silencing, cells enter precytokinesis growth arrest as a means to maintain constant cell surface VSG levels. We have investigated whether arrested cells also alter the rate of natural VSG turnover as a means to conserve the surface coat. This work provides insights into the natural biology of the glycocalyx of this important human and veterinary parasite.

Amino acid determinants of substrate selectivity in the Trypanosoma brucei sphingolipid synthase family.

The substrate selectivity of four Trypanosoma brucei sphingolipid synthases was examined. TbSLS1, an inositol phosphorylceramide (IPC) synthase, and TbSLS4, a bifunctional sphingomyelin (SM)/ethanolamine phosphorylceramide (EPC) synthase, were inactivated by Ala substitutions of a conserved triad of residues His210, His253, and Asp257 thought to form part of the active site. TbSLS4 also catalyzed the reverse reaction, production of ceramide from sphingomyelin, but none of the Ala substitutions of the catalytic triad in TbSLS4 were able to do so. Site-directed mutagenesis identified residues proximal to the conserved triad that were responsible for the discrimination between charge and size of the different head groups. For discrimination between anionic (phosphoinositol) and zwitterionic (phosphocholine, phosphoethanolamine) head groups, doubly mutated V172D/S252F TbSLS1 and D172V/F252S TbSLS3 showed reciprocal conversion between IPC and bifunctional SM/EPC synthases. For differentiation of zwitterionic headgroup size, N170A TbSLS1 and A170N/N187D TbSLS4 showed reciprocal conversion between EPC and bifunctional SM/EPC synthases. These studies provide a mapping of the SLS active site and demonstrate that differences in catalytic specificity of the T. brucei enzyme family are controlled by natural variations in as few as three residue positions.