Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites


Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.


Vyšlo v časopise: Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites. PLoS Pathog 7(10): e32767. doi:10.1371/journal.ppat.1002280
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002280

Souhrn

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.


Zdroje

1. DubeyJP 2010 Toxoplasmosis of animals and humans Boca Raton CRC Press 313

2. MillerLHGoodMFMilonG 1994 Malaria pathogenesis. Science 264 1878 1883

3. SibleyLD 1995 Invasion of vertebrate cells by Toxoplasma gondii. Trends Cell Biol 5 129 132

4. RyningFWRemingtonJS 1978 Effect of cytochalasin D on Toxoplasma gondii cell entry. Infect Immun 20 739 743

5. DobrowolskiJMSibleyLD 1996 Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 84 933 939

6. HeintzelmanMB 2006 Cellular and molecular mechanics of gliding locomotion in eukaryotes. Int Rev Cytol 251 79 129

7. HåkanssonSMorisakiHHeuserJESibleyLD 1999 Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion. Mol Biol Cell 10 3539 3547

8. VanderbergJP 1974 Studies on the motility of Plasmodium sporozoites. J Protozool 21 527 537

9. WetzelDMSchmidtJKuhlenschmidtMDubeyJPSibleyLD 2005 Gliding motility leads to active cellular invasion by Cryptosporidium parvum sporozoites. Infect Immun 73 5379 5387

10. RussellDGSindenRE 1981 The role of the cytoskeleton in the motility of coccidian sporozoites. J Cell Sci 50 345 359

11. KingCA 1981 Cell surface interaction of the protozoan Gregarina with Concanavalin A beads - implications for models of gregarine gliding. Cell Biol Intl Rep 5 297 305

12. SibleyLD 2010 How apicomplexan parasites move in and out of cells. Curr Opin Biotechnol 21 592 598

13. DobrowolskiJMNiesmanIRSibleyLD 1997 Actin in the parasite Toxoplasma gondii is encoded by a single copy gene, ACT1 and exists primarily in a globular form. Cell Motil Cytoskel 37 253 262

14. WesselingJGSmitsMASchoenmakers 1988 Extremely diverged actin proteins in Plasmodium falciparum. Mol Biochem Parasitol 30 143 154

15. WesselingJGSnijdersPJFvan SomerenPJansenJSmitsMA 1989 Stage-specific expression and genomic organization of the actin genes of the malaria parasite Plasmodium falciparum. Mol Biochem Parasitol 35 167 176

16. SchmitzSGraingerMHowellSACalderLJGaebM 2005 Malaria parasite actin filaments are very short. J Mol Biol 349 113 125

17. WetzelDMHakanssonSHuKRoosDSibleyLD 2003 Actin filament polymerization regulates gliding motility by apicomplexan parasites. Mol Biol Cell 14 396 406

18. PollardTDBlanchoinLMullinsRD 2000 Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Struct 29 545 576

19. SchülerHMuellerAKMatuschewskiK 2005 Unusual properties of Plasmodium falciparum actin: new insights into microfilament dynamics of apicomplexan parasites. FEBS Letters 579 655 660

20. BaumJPapenfussATBaumBSpeedTPCowmanAF 2006 Regulation of apicomplexan actin-based motility. Nat Rev Microbiol 4 621 628

21. SchülerHMatuschewskiK 2006 Regulation of apicomplexan microfilament dynamics by minimal set of actin-binding proteins. Traffic 7 1433 1439

22. GordonJLSibleyLD 2005 Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites. BMC Genomics 6 e179

23. SahooNBeattyWLHeuserJESeptDSibleyLD 2006 Unusual kinetic and structural properties control rapid assembly and turnover of actin in the parasite Toxoplasma gondii. Mol Biol Cell 17 895 906

24. SchmitzSSchaapIAKleinjungJHarderSGraingerM 2010 Malaria parasite actin polymerisation and filament structure. J Biol Chem 285 36577 36585

25. CrewsPManesLVBoehlerM 1986 Jasplakinolide, a cyclodepsipeptide from the marine sponge, Jaspis spp. Tetrahedron Lett 27 2797 2800

26. PoupelOTardieuxI 1999 Toxoplasma gondii motility and host cell invasiveness are drastically impaired by jasplakinolide, a cyclic peptide stabilizing F-actin. Microbes Infect 1 653 662

27. MizunoYMakiokaAKawazuSKanoSKawaiS 2002 Effect of jasplakinolide on the growth, invasion, and actin cytoskeleton of Plasmodium falciparum. Parasitol Research 88 844 848

28. Siden-KiamosIPinderJCLouisC 2006 Involvement of actin and myosins in Plasmodium berghei ookinete motility. Mol Biochem Parasitol 150 308 317

29. SmytheWAJoinerKAHoppeHC 2008 Actin is required for endocytic trafficking in the malaria parasite Plasmodium falciparum. Cell Microbiol 10 452 464

30. ShawMKTilneyLG 1999 Induction of an acrosomal process in Toxoplasma gondii: visualization of actin filaments in a protozoan parasite. Proc Natl Acad Sci U S A 96 9095 9099

31. OdaTNambaKMaedaY 2005 Position and orientation of phalloidin in F-actin determined by X-Ray fiber diffraction analysis. Biophys J 88 2727 2736

32. OdaTIwasaMAiharaTMaedaYNaritaA 2009 The nature of the globular- to fibrous-actin transition. Nature 457 441 445

33. FaulstichHZobeleySHeintzDDrewesG 1993 Probing the phalloidin binding site of actin. FEBS Letters 318 218 222

34. SteinmetzMOStofflerDMüllerSAJahnWWolpensingerB 1998 Evaluating atomic models of F-actin with an undecagold-tagged phalloidin derivative. J Mol Biol 276 1 6

35. BelmontJDPattersonGMDrubinDG 1999 New actin mutants allow further characterization of the nucleotide binding cleft and drug binding sites. J Cell Sci 112 1325 1336

36. ChenXCookRKRubensteinPA 1993 Yeast actin with a mutation in the hydrophobic plug between subdomains 3 and 4 (L266D) displays a cold-sentitive polymerization defect. J Cell Biol 123 1185 1195

37. Herm-GotzAAgop-NersesianCMunterSGrimleyJSWandlessTJ 2007 Rapid control of protein level in the apicomplexan Toxoplasma gondii. Nat Methods 4 1003 1005

38. BubbMRSpectorIBeyerBBFosenKM 2000 Effects of jasplakinolide on the kinetics of actin polymerization: an explanation for certain in vivo observations. J Biol Chem 275 5163 5170

39. McKaneMWenKKMeyerARubensteinPA 2006 Effect of the substitution of muscle actin-specific subdomain 1 and 2 residues in yeast actin on actin function. J Biol Chem 281 29916 29928

40. TiSCPollardTD 2011 Purification of actin from fission yeast Schizosaccharomyces pombe and characterization of functional differences from muscle actin. J Biol Chem 286 5784 5792

41. SchoenenbergerCABischlerNFahrenkrogBAebiU 2002 Actin's propensity for dynamic filament patterning. FEBS Letters 529 27 33

42. FujiiTIwaneAHYanagidaTNambaK 2010 Direct visualization of secondary structures of F-actin by electron cryomicroscopy. Nature 467 724 728

43. GalkinVEOrlovaASchroderGFEgelmanEH 2010 Structural polymorphism in F-actin. Nat Struct Mol Biol 17 1318 1323

44. KuangBRubensteinPA 1997 Beryllium fluoride and phalloidin retore polymerizability of a mutant yeast actin (V266G, L267G) with severely decreased hydrophobicty in a subdomain 3/4 loop. J Biol Chem 272 1237 1247

45. CowmanAFCrabbBS 2006 Invasion of red blood cells by malaria parasites. Cell 124 755 766

46. MunterSSabassBSelhuber-UnkelCKudryashevMHeggeS 2009 Plasmodium sporozoite motility is modulated by the turnover of discrete adhesion sites. Cell Host Microbe 6 551 562

47. HåkanssonSCharronAJSibleyLD 2001 Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole. Embo J 20 3132 3144

48. FrenalKSoldati-FavreD 2009 Role of the parasite and host cytoskeleton in apicomplexa parasitism. Cell Host Microbe 5 602 611

49. GaskinsEGilkSDeVoreNMannTWardGE 2004 Identification of the membrane receptor of a class XIV myosin Toxoplasma gondii. J Cell Biol 165 383 393

50. Herm-GotzAWeissSStratmannRFujita-BeckerSRuffC 2002 Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor. Embo J 21 2149 2158

51. BaumJTonkinCJPaulASRugMSmithBJ 2008 A malaria parasite formin regulates actin polymerization and localizes to the parasite-erythrocyte moving junction during invasion. Cell Host Microbe 3 188 198

52. DaherWPlattnerFCarlierMFSoldati-FavreD 2010 Concerted action of two formins in gliding motility and host cell invasion by Toxoplasma gondii. PLoS Pathog 6 e1001132

53. MehtaSSibleyLD 2011 Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii. Molec Biol Cell 22 1290 1299

54. ShawMK 2003 Cell invasion by Theileria sporozoites. Trends Parasitol 19 2 6

55. MonteroERodriguezMOksovYLoboCA 2009 Babesia divergens apical membrane antigen 1 and its interaction with the human red blood cell. Infect Immun 77 4783 4793

56. KapoorPSahasrabuddheAAKumarAMitraKSiddiqiMI 2008 An unconventional form of actin in protozoan hemoflagellate, Leishmania. J Biol Chem 283 22760 22773

57. ParedezARAssafZJSeptDTimofejevaLDawsonSC 2011 An actin cytoskeleton with evolutionaryly conserved functions in the absence of cannonical actin-binding protiens. Proc Natl Acad Sci U S A 108 6151 6156

58. HironoMKamagaiYNumataOWatanabeY 1989 Purification of Tetrahymena actin reveals some unusual properties. Proc Natl Acad Sci U S A 86 75 79

59. OtterbeinLRGraceffaPDominguezR 2001 The crysal structure of uncomplexed actin in the ADP bound state. Science 293 708 711

60. Martí-RenomMAStuartACFiserASánchezRMeloF 2000 Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct 29 291 325

61. HumphreyWDalkeASchultenK 1996 VMD: visual modeling dynamics. Graph 14 33 38

62. ZanottiGFalcignoLSavianoMD'AuriaGBrunoBM 2001 Solid state and solution conformation of [Ala7]-phalloidin: a synthetic phallotoxin analogue. Chemistry 7 1479 1485

63. KaleLSkeelRBhandarkarMBrunnerRGursoyA 1999 NAMD2: Greater scalability for parallel molecular dynamics. J Comp Physics 151 283 312

64. MorrisGMGoodsellDSHallidayRSHueyRHartWE 1998 Automated docking using a Lamarkian genetic algorithm and emperical binding free energy function. J Comp Chem 19 1639 1662

65. MorisakiJHHeuserJESibleyLD 1995 Invasion of Toxoplasma gondii occurs by active penetration of the host cell. J Cell Sci 108 2457 2464

66. LouridoSShumanJZhangCShokatKMHuiR 2010 Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature 465 359 362

67. HigginsDGThompsonJDGibsonTJ 1996 Using CLUSTAL for multiple sequence alignments. Methods Enzymol 266 382 402

68. SwoffordDL 2002 Phylogenetic Analysis Using Parsimony (* and other methods) Sunderland Sinauer Associates

69. PageRD 2002 Visualizing phylogenetic trees using TreeView. Curr Protoc Bioinformatics Chapter 6 Unit 6 2

70. PondSLFrostSDMuseSV 2005 HyPhy: hypothesis testing using phylogenies. Bioinform 21 676 679

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2011 Číslo 10
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa