RESUMO
Leishmaniasis, a vector-borne disease, is caused by the infection of Leishmania spp., obligate intracellular protozoan parasites. Presently, human vaccines are unavailable, and the primary treatment relies heavily on systemic drugs, often presenting with suboptimal formulations and substantial toxicity, making new drugs a high priority for LMIC countries burdened by the disease, but a low priority in the agenda of most pharmaceutical companies due to unattractive profit margins. New ways to accelerate the discovery of new, or the repositioning of existing drugs, are needed. To address this challenge, our study aimed to identify potential protein targets shared among clinically-relevant Leishmania species. We employed a subtractive proteomics and comparative genomics approach, integrating high-throughput multi-omics data to classify these targets based on different druggability metrics. This effort resulted in the ranking of 6502 ortholog groups of protein targets across 14 pathogenic Leishmania species. Among the top 20 highly ranked groups, metabolic processes known to be attractive drug targets, including the ubiquitination pathway, aminoacyl-tRNA synthetases, and purine synthesis, were rediscovered. Additionally, we unveiled novel promising targets such as the nicotinate phosphoribosyltransferase enzyme and dihydrolipoamide succinyltransferases. These groups exhibited appealing druggability features, including less than 40% sequence identity to the human host proteome, predicted essentiality, structural classification as highly druggable or druggable, and expression levels above the 50th percentile in the amastigote form. The resources presented in this work also represent a comprehensive collection of integrated data regarding trypanosomatid biology.
Assuntos
Antiprotozoários , Leishmania , Leishmaniose , Proteômica , Proteínas de Protozoários , Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Leishmania/genética , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Humanos , Descoberta de Drogas , GenômicaRESUMO
Visceral leishmaniasis and Chagas disease are neglected tropical diseases (NTDs) that severely impact the developing world. With current therapies suffering from poor efficacy and safety profiles as well as emerging resistance, new drug leads are direly needed. In this work, 26 alkaloids (9 natural and 17 synthetic) belonging to the benzyltetrahydroisoquinoline (BI) family were evaluated against both the pro/trypomastigote and amastigote forms of the parasites Leishmania infantum and Trypanosoma cruzi, the causative agents of these diseases. These alkaloids were synthesized via an efficient and modular enantioselective approach based on Bischler-Napieralski cyclization/Noyori asymmetric transfer hydrogenation to build the tetrahydroisoquinoline core. The bis-benzyltetrahydroisoquinoline (BBI) alkaloids were prepared using an Ullmann coupling of two BI units to form the biaryl ether linkage, which enabled a comprehensive survey of the influence of BI stereochemistry on bioactivity. Preliminary studies into the mechanism of action against Leishmania mexicana demonstrate that these compounds interfere with the cell cycle, potentially through inhibition of kinetoplast division, which may offer opportunities to identify a new target/mechanism of action. Three of the synthesized alkaloids showed promising druglike potential, meeting the Drugs for Neglected Disease initiative (DNDi) criteria for a hit against Chagas disease.
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Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The '9+2' axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked whether there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana have an extensive RSP complement, including two divergent RSP9 orthologues, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither orthologue is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 orthologue, deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked to the different mode of assembly of trypanosomatid versus Plasmodium flagella.
Assuntos
Parasitos , Plasmodium , Masculino , Animais , Axonema/metabolismo , Parasitos/metabolismo , Microtúbulos/metabolismo , Sementes , Proteínas/metabolismo , Flagelos/metabolismo , Eucariotos/metabolismo , Plasmodium/metabolismo , Dineínas/metabolismoRESUMO
Background: Genome-wide subcellular protein localisation in Trypanosoma brucei, through our TrypTag project, has comprehensively dissected the molecular organisation of this important pathogen. Powerful as this resource is , T. brucei has multiple developmental forms and we previously only analysed the procyclic form. This is an insect life cycle stage, leaving the mammalian bloodstream form unanalysed. The expectation is that between life stages protein localisation would not change dramatically (completely unchanged or shifting to analogous stage-specific structures). However, this has not been specifically tested. Similarly, which organelles tend to contain proteins with stage-specific expression can be predicted from known stage specific adaptations but has not been comprehensively tested. Methods: We used endogenous tagging with mNG to determine the sub-cellular localisation of the majority of proteins encoded by transcripts significantly upregulated in the bloodstream form, and performed comparison to the existing localisation data in procyclic forms. Results: We have confirmed the localisation of known stage-specific proteins and identified the localisation of novel stage-specific proteins. This gave a map of which organelles tend to contain stage specific proteins: the mitochondrion for the procyclic form, and the endoplasmic reticulum, endocytic system and cell surface in the bloodstream form. Conclusions: This represents the first genome-wide map of life cycle stage-specific adaptation of organelle molecular machinery in T. brucei.
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We present the genome sequence of Leishmania mexicana MNYC/BZ/62/M379 modified to express Cas9 and T7 RNA-polymerase, revealing high similarity to the reference genome (MHOM/GT2001/U1103). Through RNAseq-based annotation of coding sequences and untranslated regions, we provide primer sequences for construct and sgRNA template generation for CRISPR-assisted gene deletion and endogenous tagging.
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Trypanosomes and related parasites such as Leishmania are unicellular parasites with a precise internal structure. This makes light microscopy a powerful tool for interrogating their biology-whether considering advance techniques for visualizing the precise localization of proteins within the cell or simply measuring parasite cell shape. Methods to partially or fully automate analysis and interpretation are extremely powerful and provide easier access to microscope images as a source of quantitative data. This chapter provides an introduction to these methods using ImageJ/FIJI, free and open source software for scientific image analysis. It provides an overview of how ImageJ handles images and introduces the ImageJ macro/scripting language for automated images, starting at a basic level and assuming no previous programming/scripting experience. It then outlines three methods using ImageJ for automated analysis of trypanosome micrographs: Semiautomated cropping and setting image contrast for presentation, automated analysis of cell properties from a light micrograph field of view, and example semiautomated tools for quantitative analysis of protein localization. These are not presented as strict methods, but are instead described in detail with the intention of furnishing the reader with the ability to "hack" the scripts for their own needs or write their own scripts for partially and fully automated quantitation of trypanosomes from light micrographs. Most of the methods described here are transferrable to other types of microscope image and other cell types.
Assuntos
Processamento de Imagem Assistida por Computador , Software , Trypanosoma/citologia , Núcleo Celular , DNA de Cinetoplasto/análise , Microscopia/métodosRESUMO
Eukaryotic flagella are conserved multifunctional organelles with roles in motility, intercellular interactions, and signal transduction. Leishmania possess a single flagellum at all stages of their life cycle. Flagella of promastigote forms in the fly are long and motile, with a canonical 9 + 2 microtubule axoneme and an extra-axonemal paraflagellar rod (PFR). This protocol describes a simple method for the isolation of Leishmania mexicana promastigote flagella, optimized to yield intact flagella that retain both the cytoskeletal elements (9 + 2 axoneme and PFR) and the surrounding membrane. The isolated flagella and deflagellated cell bodies are suitable for analysis by electron microscopy, protein mass spectrometry, and lipidomics.
Assuntos
Fracionamento Celular/métodos , Flagelos/metabolismo , Leishmania mexicana/citologia , Estágios do Ciclo de Vida , Centrifugação com Gradiente de Concentração , Citoesqueleto/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Lipidômica , Espectrometria de Massas , Microscopia Eletrônica , Proteínas de Protozoários/análise , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismoRESUMO
The Leishmania lysosome has an atypical structure, consisting of an elongated vesicle-filled tubule running along the anterior-posterior axis of the cell, which is termed the multivesicular tubule (MVT) lysosome. Alongside, the MVT lysosome is one or more microtubules, the lysosomal microtubule(s). Previous work indicated there were cell cycle-related changes in MVT lysosome organization; however, these only provided snapshots and did not connect the changes in the lysosomal microtubule(s) or lysosomal function. Using mNeonGreen tagged cysteine peptidase A and SPEF1 as markers of the MVT lysosome and lysosomal microtubule(s), we examined the dynamics of these structures through the cell cycle. Both the MVT lysosome and lysosomal microtubule(s) elongated at the beginning of the cell cycle before plateauing and then disassembling in late G2 before cytokinesis. Moreover, the endocytic rate in cells where the MVT lysosome and lysosomal microtubule(s) had disassembled was extremely low. The dynamic nature of the MVT lysosome and lysosomal microtubule(s) parallels that of the Trypanosoma cruzi cytostome/cytopharynx, which also has a similar membrane tubule structure with associated microtubules. As the cytostome/cytopharynx is an ancestral feature of the kinetoplastids, this suggests that the Leishmania MVT lysosome and lysosomal microtubule(s) are a reduced cytostome/cytopharynx-like feature.
Assuntos
Endocitose , Interações Hospedeiro-Parasita , Leishmania/fisiologia , Leishmaniose/metabolismo , Leishmaniose/parasitologia , Lisossomos/parasitologia , Divisão Celular , Citocinese , Flagelos , Leishmania/ultraestrutura , Leishmaniose/imunologiaRESUMO
Trypanosomes have complex life cycles within which there are both proliferative and differentiation cell divisions. The coordination of the cell cycle to achieve these different divisions is critical for the parasite to infect both host and vector. From studying the regulation of the proliferative cell cycle of the Trypanosoma brucei procyclic life cycle stage, three subcycles emerge that control the duplication and segregation of (a) the nucleus, (b) the kinetoplast, and (c) a set of cytoskeletal structures. We discuss how the clear dependency relationships within these subcycles, and the potential for cross talk between them, are likely required for overall cell cycle coordination. Finally, we look at the implications this interdependence has for proliferative and differentiation divisions through the T. brucei life cycle and in related parasitic trypanosomatid species.
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Ciclo Celular , Trypanosoma brucei brucei/crescimento & desenvolvimento , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , DNA de Cinetoplasto/metabolismo , DNA de Protozoário/metabolismo , Regulação da Expressão GênicaRESUMO
Analysis of flagellum and cilium beating in three dimensions (3D) is important for understanding cell motility, and using fluorescence microscopy to do so would be extremely powerful. Here, high-speed multifocal plane fluorescence microscopy, where the light path is split to visualise multiple focal planes simultaneously, was used to reconstruct Trypanosoma brucei and Leishmania mexicana movement in 3D. These species are uniflagellate unicellular parasites for which motility is vital. It was possible to use either a fluorescent stain or a genetically-encoded fluorescent protein to visualise flagellum and cell movement at 200â Hz frame rates. This addressed two open questions regarding Trypanosoma and Leishmania flagellum beating, which contributes to their swimming behaviours: 1) how planar is the L. mexicana flagellum beat, and 2) what is the nature of flagellum beating during T. brucei 'tumbling'? We showed that L. mexicana has notable deviations from a planar flagellum beat, and that during tumbling the T. brucei flagellum bends the cell and beats only in the distal portion to achieve cell reorientation. This demonstrates high-speed multifocal plane fluorescence microscopy as a powerful tool for the analysis of beating flagella.
Assuntos
Flagelos , Leishmania mexicana/citologia , Movimento (Física) , Trypanosoma brucei brucei/citologia , Microscopia de Fluorescência , Microscopia de VídeoRESUMO
Differentiation of Trypanosoma brucei, a flagellated protozoan parasite, between life cycle stages typically occurs through an asymmetric cell division process, producing two morphologically distinct daughter cells. Conversely, proliferative cell divisions produce two daughter cells, which look similar but are not identical. To examine in detail differences between the daughter cells of a proliferative division of procyclic T. brucei we used the recently identified constituents of the flagella connector. These segregate asymmetrically during cytokinesis allowing the new-flagellum and the old-flagellum daughters to be distinguished. We discovered that there are distinct morphological differences between the two daughters, with the new-flagellum daughter in particular re-modelling rapidly and extensively in early G1. This re-modelling process involves an increase in cell body, flagellum and flagellum attachment zone length and is accompanied by architectural changes to the anterior cell end. The old-flagellum daughter undergoes a different G1 re-modelling, however, despite this there was no difference in G1 duration of their respective cell cycles. This work demonstrates that the two daughters of a proliferative division of T. brucei are non-equivalent and enables more refined morphological analysis of mutant phenotypes. We suggest all proliferative divisions in T. brucei and related organisms will involve non-equivalence.
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Flagelos/metabolismo , Trypanosoma brucei brucei/citologia , Divisão Celular , Proliferação de Células , Citocinese , Flagelos/genética , Estágios do Ciclo de Vida , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismoRESUMO
Ubiquitous in eukaryotic organisms, the flagellum is a well-studied organelle that is well-known to be responsible for motility in a variety of organisms. Commonly necessitated in their study is the capability to image and subsequently track the movement of one or more flagella using videomicroscopy, requiring digital isolation and location of the flagellum within a sequence of frames. Such a process in general currently requires some researcher input, providing some manual estimate or reliance on an experiment-specific heuristic to correctly identify and track the motion of a flagellum. Here we present a fully-automated method of flagellum identification from videomicroscopy based on the fact that the flagella are of approximately constant width when viewed by microscopy. We demonstrate the effectiveness of the algorithm by application to captured videomicroscopy of Leishmania mexicana, a parasitic monoflagellate of the family Trypanosomatidae. ImageJ Macros for flagellar identification are provided, and high accuracy and remarkable throughput are achieved via this unsupervised method, obtaining results comparable in quality to previous studies of closely-related species but achieved without the need for precursory measurements or the development of a specialised heuristic, enabling in general the automated generation of digitised kinematic descriptions of flagellar beating from videomicroscopy.
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Movimento Celular/fisiologia , Flagelos/ultraestrutura , Leishmania mexicana/ultraestrutura , Microscopia de Vídeo , Fenômenos Biomecânicos , Flagelos/fisiologia , Humanos , Leishmania mexicana/patogenicidade , Leishmania mexicana/fisiologiaRESUMO
NMR and MRI diffusion experiments contain information describing the shape, size, abundance, and membrane permeability of cells although extracting this information can be challenging. Here we present the INDIANA (IN-cell DIffusion ANAlysis) method to simultaneously and non-invasively measure cell abundance, effective radius, permeability and intrinsic relaxation rates and diffusion coefficients within the inter- and intra-cellular populations. The method couples an experimental dataset comprising stimulated-echo diffusion measurements, varying both the gradient strength and the diffusion delay, together with software to fit a model based on the Kärger equations to robustly extract the relevant parameters. A detailed error analysis is presented by comparing the results from fitting simulated data from Monte Carlo simulations, establishing its effectiveness. We note that for parameters typical of mammalian cells the approach is particularly effective, and the shape of the underlying cells does not unduly affect the results. Finally, we demonstrate the performance of the experiment on systems of suspended yeast and mammalian cells. The extracted parameters describing cell abundance, size, permeability and relaxation are independently validated.
Assuntos
Permeabilidade da Membrana Celular , Tamanho Celular , Células/ultraestrutura , Imagem de Difusão por Ressonância Magnética/métodos , Algoritmos , Animais , Linhagem Celular , Simulação por Computador , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Método de Monte Carlo , Software , Leveduras/ultraestruturaRESUMO
It is well established that the parasites of the genus Leishmania exhibit complex surface interactions with the sandfly vector midgut epithelium, but no prior study has considered the details of their hydrodynamics. Here, the boundary behaviours of motile Leishmania mexicana promastigotes are explored in a computational study using the boundary element method, with a model flagellar beating pattern that has been identified from digital videomicroscopy. In particular a simple flagellar kinematics is observed and quantified using image processing and mode identification techniques, suggesting a simple mechanical driver for the Leishmania beat. Phase plane analysis and long-time simulation of a range of Leishmania swimming scenarios demonstrate an absence of stable boundary motility for an idealised model promastigote, with behaviours ranging from boundary capture to deflection into the bulk both with and without surface forces between the swimmer and the boundary. Indeed, the inclusion of a short-range repulsive surface force results in the deflection of all surface-bound promastigotes, suggesting that the documented surface detachment of infective metacyclic promastigotes may be the result of their particular morphology and simple hydrodynamics. Further, simulation elucidates a remarkable morphology-dependent hydrodynamic mechanism of boundary approach, hypothesised to be the cause of the well-established phenomenon of tip-first epithelial attachment of Leishmania promastigotes to the sandfly vector midgut.
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Hidrodinâmica , Leishmania mexicana/fisiologia , Psychodidae/parasitologia , Animais , Fenômenos Biofísicos , Insetos Vetores/parasitologia , Estágios do Ciclo de Vida , NataçãoRESUMO
Compartmentalization is a characterizing feature of complexity in cells, used to organize their biochemistry. Membrane-bound organelles are most widely known, but non-membrane-bound liquid organelles also exist. These have recently been shown to form by phase separation of specific types of proteins known as scaffolds. This forms two phases: a condensate that is enriched in scaffold protein separated by a phase boundary from the cytoplasm or nucleoplasm with a low concentration of the scaffold protein. Phase separation is well known for synthetic polymers, but also appears important in cells. Here, we review the properties of proteins important for forming these non-membrane-bound organelles, focusing on the energetically favourable interactions that drive condensation. On this basis we make qualitative predictions about how cells may control compartmentalization by condensates; the partition of specific molecules to a condensate; the control of condensation and dissolution of condensates; and the regulation of condensate nucleation. There are emerging data supporting many of these predictions, although future results may prove incorrect. It appears that many molecules may have the ability to modulate condensate formation, making condensates a potential target for future therapeutics. The emerging properties of condensates are fundamentally unlike the properties of membrane-bound organelles. They have the capacity to rapidly integrate cellular events and act as a new class of sensors for internal and external environments.This article is part of the theme issue 'Self-organization in cell biology'.
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Organelas/metabolismo , Proteínas/química , Fenômenos Biofísicos , CitoplasmaRESUMO
We describe a single-step centrifugal elutriation method to produce synchronous Gap1 (G1)-phase procyclic trypanosomes at a scale amenable for proteomic analysis of the cell cycle. Using ten-plex tandem mass tag (TMT) labeling and mass spectrometry (MS)-based proteomics technology, the expression levels of 5325 proteins were quantified across the cell cycle in this parasite. Of these, 384 proteins were classified as cell-cycle regulated and subdivided into nine clusters with distinct temporal regulation. These groups included many known cell cycle regulators in trypanosomes, which validates the approach. In addition, we identify 40 novel cell cycle regulated proteins that are essential for trypanosome survival and thus represent potential future drug targets for the prevention of trypanosomiasis. Through cross-comparison to the TrypTag endogenous tagging microscopy database, we were able to validate the cell-cycle regulated patterns of expression for many of the proteins of unknown function detected in our proteomic analysis. A convenient interface to access and interrogate these data is also presented, providing a useful resource for the scientific community. Data are available via ProteomeXchange with identifier PXD008741 (https://www.ebi.ac.uk/pride/archive/).
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Ciclo Celular/fisiologia , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , ProteômicaRESUMO
Microscale swimming may be intuited to be dominated by background flows, sweeping away any untethered bodies with the prevalent flow direction. However, it has been observed that many microswimmers utilize ambient flows as guidance cues, in some cases resulting in net motion upstream, contrary to the dominant background fluid direction and our accompanying intuition. Thus the hydrodynamic response of small-scale motile organisms requires careful analysis of the complex interaction between swimmer and environment. Here we investigate the effects of a Newtonian shear flow on monoflagellated swimmers with specified body symmetry, representing, for instance, the Leishmania mexicana promastigote, a parasitic hydrodynamic puller that inhabits the microenvironment of a sandfly vector midgut and is the cause of a major and neglected human tropical disease. We observe that a lack of symmetry-breaking cellular geometry results in the periodic tumbling of swimmers in the bulk, with the rotations exhibiting a linear response to changes in shearing rate enabling analytic approximation. In order to draw comparisons with the better-studied pushers, we additionally consider virtual Leishmania promastigotes in a confined but typical geometry, that of a no-slip planar solid boundary, and note that in general stable guided taxis is not exhibited amongst the range of observed behaviors. However, a repulsive boundary gives rise to significant continued taxis in the presence of shearing flow, a phenomenon that may be of particular pertinence to the infective lifecycle stage of such swimmers subject to the assumption of a Newtonian medium. We finally propose a viable and general in vitro method of controlling microswimmer boundary accumulation using temporally evolving background shear flows, based on the analysis of phase-averaged dynamics and verified in silico.
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Flagelos/metabolismo , Hidrodinâmica , Fenômenos Mecânicos , Leishmania/citologia , Leishmania/fisiologia , Modelos Moleculares , NataçãoRESUMO
Paratrypanosoma confusum is a monoxenous kinetoplastid flagellate that constitutes the most basal branch of the highly diverse parasitic trypanosomatids, which include human pathogens Trypanosoma and Leishmania This makes Paratrypanosoma uniquely informative for the evolution of obligatory parasitism from free-living lifestyle and the evolution of human parasitism in some trypanosomatid lineages. It has typical promastigote morphology but also forms surface-attached haptomonads and amastigotes. Haptomonads form by attachment to a surface via a large bulge at the base of the flagellum, which is then remodeled into a thin attachment pad associated with flagellum shortening. Promastigotes and haptomonads multiply by binary division, and the progeny of a haptomonad can either remain attached or grow a flagellum and resume swimming. Whole genome sequencing and transcriptome profiling, in combination with analysis of the cell ultrastructure, reveal how the cell surface and metabolism are adapted to parasitism and how characteristic cytoskeletal features are conserved. Our data demonstrate that surface attachment by the flagellum and the flagellar pocket, a Leishmania-like flagellum attachment zone, and a Trypanosoma cruzi-like cytostome are ancestral features, while evolution of extant trypanosomatids, including the human parasites, is associated with genome streamlining and diversification of membrane proteins.
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Flagelos/genética , Estágios do Ciclo de Vida/genética , Trypanosoma cruzi/genética , Citoesqueleto/genética , Perfilação da Expressão Gênica/métodos , Genoma de Protozoário/genética , Humanos , Leishmania/genética , Filogenia , Proteínas de Protozoários/genéticaRESUMO
The extracellular bloodstream form parasite Trypanosoma brucei is supremely adapted to escape the host innate and adaptive immune system. Evasion is mediated through an antigenically variable Variant Surface Glycoprotein (VSG) coat, which is recycled at extraordinarily high rates. Blocking VSG synthesis triggers a precytokinesis arrest where stalled cells persist for days in vitro with superficially intact VSG coats, but are rapidly cleared within hours in mice. We therefore investigated the role of VSG synthesis in trypanosome phagocytosis by activated mouse macrophages. T. brucei normally effectively evades macrophages, and induction of VSG RNAi resulted in little change in phagocytosis of the arrested cells. Halting VSG synthesis resulted in stalled cells which swam directionally rather than tumbling, with a significant increase in swim velocity. This is possibly a consequence of increased rigidity of the cells due to a restricted surface coat in the absence of VSG synthesis. However if VSG RNAi was induced in the presence of anti-VSG221 antibodies, phagocytosis increased significantly. Blocking VSG synthesis resulted in reduced clearance of anti-VSG antibodies from the trypanosome surface, possibly as a consequence of the changed motility. This was particularly marked in cells in the G2/ M cell cycle stage, where the half-life of anti-VSG antibody increased from 39.3 ± 4.2 seconds to 99.2 ± 15.9 seconds after induction of VSG RNAi. The rates of internalisation of bulk surface VSG, or endocytic markers like transferrin, tomato lectin or dextran were not significantly affected by the VSG synthesis block. Efficient elimination of anti-VSG-antibody complexes from the trypanosome cell surface is therefore essential for trypanosome evasion of macrophages. These experiments highlight the essentiality of high rates of VSG recycling for the rapid removal of host opsonins from the parasite surface, and identify this process as a key parasite virulence factor during a chronic infection.