High Affinity Nanobodies against the VSG Are Potent Trypanolytic Agents that Block Endocytosis

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The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment.

Vyšlo v časopise: High Affinity Nanobodies against the VSG Are Potent Trypanolytic Agents that Block Endocytosis. PLoS Pathog 7(6): e32767. doi:10.1371/journal.ppat.1002072
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002072

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1. BarrettMPBurchmoreRJStichALazzariJOFraschAC 2003 The trypanosomiases. Lancet 362 1469 1480

2. SternbergJM 2004 Human African trypanosomiasis: clinical presentation and immune response. Parasite Immunol 26 469 476

3. DonelsonJEHillKLEl-SayedNM 1998 Multiple mechanisms of immune evasion by African trypanosomes. Mol Biochem Parasitol 91 51 66

4. VanhammeLPaysEMcCullochRBarryJD 2001 An update on antigenic variation in African trypanosomes. Trends Parasitol 17 338 343

5. JacksonDGOwenMJVoorheisHP 1985 A new method for the rapid purification of both the membrane-bound and released forms of the variant surface glycoprotein from Trypanosoma brucei. Biochem J 230 195 202

6. BarryJDMcCullochR 2001 Antigenic variation in trypanosomes: enhanced phenotypic variation in a eukaryotic parasite. Adv Parasitol 49 1 70

7. RussoDCWilliamsDJGrabDJ 1994 Mechanisms for the elimination of potentially lytic complement-fixing variable surface glycoprotein antibody-complexes in Trypanosoma brucei. Parasitol Res 80 487 492

8. PalAHallBSJeffriesTRFieldMC 2003 Rab5 and Rab11 mediate transferrin and anti-variant surface glycoprotein antibody recycling in Trypanosoma brucei. Biochem J 374 443 451

9. WebsterPRussoDCBlackSJ 1990 The interaction of Trypanosoma brucei with antibodies to variant surface glycoproteins. J Cell Sci 96 Pt 2 249 255

10. BalberAEBangsJDJonesSMProiaRL 1979 Inactivation or elimination of potentially trypanolytic, complement-activating immune complexes by pathogenic trypanosomes. Infect Immun 24 617 627

11. O'BeirneCLowryCMVoorheisHP 1998 Both IgM and IgG anti-VSG antibodies initiate a cycle of aggregation-disaggregation of bloodstream forms of Trypanosoma brucei without damage to the parasite. Mol Biochem Parasitol 91 165 193

12. EngstlerMPfohlTHerminghausSBoshartMWiegertjesG 2007 Hydrodynamic flow-mediated protein sorting on the cell surface of trypanosomes. Cell 131 505 515

13. FieldMCCarringtonM 2009 The trypanosome flagellar pocket. Nat Rev Microbiol 7 775 786

14. FrevertUReinwaldE 1990 Trypanosoma congolense bloodstream forms evade complement lysis in vitro by shedding of immune complexes. Eur J Cell Biol 52 264 269

15. TakayanagiTKawaguchiHYabuYItohMYanoK 1991 Dissociation of IgG antibody-mediated clumps of Trypanosoma brucei gambiense by complement. Parasitol Res 77 645 650

16. RalstonKSKabututuZPMelehaniJHOberholzerMHillKL 2009 The Trypanosoma brucei flagellum: moving parasites in new directions. Annu Rev Microbiol 63 335 362

17. GrunfelderCGEngstlerMWeiseFSchwarzHStierhofYD 2002 Accumulation of a GPI-anchored protein at the cell surface requires sorting at multiple intracellular levels. Traffic 3 547 559

18. GullK 2003 Host-parasite interactions and trypanosome morphogenesis: a flagellar pocketful of goodies. Curr Opin Microbiol 6 365 370

19. ThiloL 1985 Quantification of endocytosis-derived membrane traffic. Biochim Biophys Acta 822 243 266

20. EngstlerMThiloLWeiseFGrunfelderCGSchwarzH 2004 Kinetics of endocytosis and recycling of the GPI-anchored variant surface glycoprotein in Trypanosoma brucei. J Cell Sci 117 1105 1115

21. KabiriMSteverdingD 2000 Studies on the recycling of the transferrin receptor in Trypanosoma brucei using an inducible gene expression system. Eur J Biochem 267 3309 3314

22. OverathPEngstlerM 2004 Endocytosis, membrane recycling and sorting of GPI-anchored proteins: Trypanosoma brucei as a model system. Mol Microbiol 53 735 744

23. GrunfelderCGEngstlerMWeiseFSchwarzHStierhofYD 2003 Endocytosis of a glycosylphosphatidylinositol-anchored protein via clathrin-coated vesicles, sorting by default in endosomes, and exocytosis via RAB11-positive carriers. Mol Biol Cell 14 2029 2040

24. JeffriesTRMorganGWFieldMC 2001 A developmentally regulated rab11 homologue in Trypanosoma brucei is involved in recycling processes. J Cell Sci 114 2617 2626

25. BrighouseADacksJBFieldMC 2010 Rab protein evolution and the history of the eukaryotic endomembrane system. Cell Mol Life Sci 67 3449 3465

26. PalAHallBSNesbethDNFieldHIFieldMC 2002 Differential endocytic functions of Trypanosoma brucei Rab5 isoforms reveal a glycosylphosphatidylinositol-specific endosomal pathway. J Biol Chem 277 9529 9539

27. Hamers-CastermanCAtarhouchTMuyldermansSRobinsonGHamersC 1993 Naturally occurring antibodies devoid of light chains. Nature 363 446 448

28. MuyldermansS 2001 Single domain camel antibodies: current status. J Biotechnol 74 277 302

29. SaerensDGhassabehGHMuyldermansS 2008 Single-domain antibodies as building blocks for novel therapeutics. Curr Opin Pharmacol 8 600 608

30. StijlemansBConrathKCortez-RetamozoVVan XongHWynsL 2004 Efficient targeting of conserved cryptic epitopes of infectious agents by single domain antibodies. African trypanosomes as paradigm. J Biol Chem 279 1256 1261

31. BaralTNMagezSStijlemansBConrathKVanhollebekeB 2006 Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor. Nat Med 12 580 584

32. AllenCLGouldingDFieldMC 2003 Clathrin-mediated endocytosis is essential in Trypanosoma brucei. Embo J 22 4991 5002

33. SeyfangAMeckeDDuszenkoM 1990 Degradation, recycling, and shedding of Trypanosoma brucei variant surface glycoprotein. J Protozool 37 546 552

34. Ter KuileBHWiemerEAMichelsPAOpperdoesFR 1992 The electrochemical proton gradient in the bloodstream form of Trypanosoma brucei is dependent on the temperature. Mol Biochem Parasitol 55 21 27

35. BarrettMPTetaudESeyfangABringaudFBaltzT 1998 Trypanosome glucose transporters. Mol Biochem Parasitol 91 195 205

36. MilettiLCKoerichLBPachecoLKSteindelMStambukBU 2006 Characterization of D-glucose transport in Trypanosoma rangeli. Parasitology 133 721 727

37. WilckeMJohannesLGalliTMayauVGoudB 2000 Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network. J Cell Biol 151 1207 1220

38. HallBAllenCLGouldingDFieldMC 2004 Both of the Rab5 subfamily small GTPases of Trypanosoma brucei are essential and required for endocytosis. Mol Biochem Parasitol 138 67 77

39. DelafosseADoutoumAA 2004 Prevalence of Trypanosoma evansi infection and associated risk factors in camels in eastern Chad. Vet Parasitol 119 155 164

40. DesmyterASpinelliSPayanFLauwereysMWynsL 2002 Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology. J Biol Chem 277 23645 23650

41. TransueTRDe GenstEGhahroudiMAWynsLMuyldermansS 1998 Camel single-domain antibody inhibits enzyme by mimicking carbohydrate substrate. Proteins 32 515 522

42. StockwinLHHolmesS 2003 Antibodies as therapeutic agents: vive la renaissance! Expert Opin Biol Ther 3 1133 1152

43. NevinskyGABunevaVN 2003 Catalytic antibodies in healthy humans and patients with autoimmune and viral diseases. J Cell Mol Med 7 265 276

44. WentworthPJrJonesLHWentworthADZhuXLarsenNA 2001 Antibody catalysis of the oxidation of water. Science 293 1806 1811

45. DattaDVaidehiNXuXGoddardWA3rd 2002 Mechanism for antibody catalysis of the oxidation of water by singlet dioxygen. Proc Natl Acad Sci U S A 99 2636 2641

46. WentworthADJonesLHWentworthPJrJandaKDLernerRA 2000 Antibodies have the intrinsic capacity to destroy antigens. Proc Natl Acad Sci U S A 97 10930 10935

47. HallBSSmithELangerWJacobsLAGouldingD 2005 Developmental variation in Rab11-dependent trafficking in Trypanosoma brucei. Eukaryot Cell 4 971 980

48. LorgerMEngstlerMHomannMGoringerHU 2003 Targeting the variable surface of African trypanosomes with variant surface glycoprotein-specific, serum-stable RNA aptamers. Eukaryot Cell 2 84 94

49. McGwireBSOlsonCLTackBFEngmanDM 2003 Killing of African trypanosomes by antimicrobial peptides. J Infect Dis 188 146 152

50. DelgadoMAndersonPGarcia-SalcedoJACaroMGonzalez-ReyE 2009 Neuropeptides kill African trypanosomes by targeting intracellular compartments and inducing autophagic-like cell death. Cell Death Differ 16 406 416

51. HainesLRThomasJMJacksonAMEyfordBARazaviM 2009 Killing of trypanosomatid parasites by a modified bovine host defense peptide, BMAP-18. PLoS Negl Trop Dis 3 e373

52. NatesanSKPeacockLLeungKFGibsonWFieldMC 2010 Evidence that low endocytic activity is not directly responsible for human serum resistance in the insect form of African trypanosomes. BMC Res Notes 3 63

53. BroadheadRDaweHRFarrHGriffithsSHartSR 2006 Flagellar motility is required for the viability of the bloodstream trypanosome. Nature 440 224 227

54. ConrathKELauwereysMGalleniMMatagneAFrereJM 2001 Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. Antimicrob Agents Chemother 45 2807 2812

55. HmilaIAbdallahRBSaerensDBenlasfarZConrathK 2008 VHH, bivalent domains and chimeric Heavy chain-only antibodies with high neutralizing efficacy for scorpion toxin AahI'. Mol Immunol 45 3847 3856

56. NatesanSKPeacockLMatthewsKGibsonWFieldMC 2007 Activation of endocytosis as an adaptation to the mammalian host by trypanosomes. Eukaryot Cell 6 2029 2037

57. SaerensDFrederixFReekmansGConrathKJansK 2005 Engineering camel single-domain antibodies and immobilization chemistry for human prostate-specific antigen sensing. Anal Chem 77 7547 7555

58. FieldMCAllenCLDhirVGouldingDHallBS 2004 New approaches to the microscopic imaging of Trypanosoma brucei. Microsc Microanal 10 621 636

59. BayeleHK 2001 Triazinyl derivatives that are potent inhibitors of glucose transport in Trypanosoma brucei. Parasitol Res 87 911 914

60. BradfordMM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 248 254