Flagellar Motility Is Not Directly Required to Maintain Attachment to Surfaces
Giardia trophozoites attach to the intestinal microvilli (or inert surfaces) using an undefined “suction-based” mechanism, and remain attached during cell division to avoid peristalsis. Flagellar motility is a key factor in Giardia's pathogenesis and colonization of the host small intestine. Specifically, the beating of the ventral flagella, one of four pairs of motile flagella, has been proposed to generate a hydrodynamic force that results in suction-based attachment via the adjacent ventral disc. We aimed to test this prevailing “hydrodynamic model” of attachment mediated by flagellar motility. We defined four distinct stages of attachment by assessing surface contacts of the trophozoite with the substrate during attachment using TIRF microscopy (TIRFM). The lateral crest of the ventral disc forms a continuous perimeter seal with the substrate, a cytological indication that trophozoites are fully attached. Using trophozoites with two types of molecularly engineered defects in flagellar beating, we determined that neither ventral flagellar beating, nor any flagellar beating, is necessary for the maintenance of attachment. Following a morpholino-based knockdown of PF16, a central pair protein, both the beating and morphology of flagella were defective, but trophozoites could still initiate proper surface contacts as seen using TIRFM and could maintain attachment in several biophysical assays. Trophozoites with impaired motility were able to attach as well as motile cells. We also generated a strain with defects in the ventral flagellar waveform by overexpressing a dominant negative form of alpha2-annexin::GFP (D122A, D275A). This dominant negative alpha2-annexin strain could initiate attachment and had only a slight decrease in the ability to withstand normal and shear forces. The time needed for attachment did increase in trophozoites with overall defective flagellar beating, however. Thus while not directly required for attachment, flagellar motility is important for positioning and orienting trophozoites prior to attachment. Drugs affecting flagellar motility may result in lower levels of attachment by indirectly limiting the number of parasites that can position the ventral disc properly against a surface and against peristaltic flow.
Vyšlo v časopise:
Flagellar Motility Is Not Directly Required to Maintain Attachment to Surfaces. PLoS Pathog 7(8): e32767. doi:10.1371/journal.ppat.1002167
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.ppat.1002167
Souhrn
Giardia trophozoites attach to the intestinal microvilli (or inert surfaces) using an undefined “suction-based” mechanism, and remain attached during cell division to avoid peristalsis. Flagellar motility is a key factor in Giardia's pathogenesis and colonization of the host small intestine. Specifically, the beating of the ventral flagella, one of four pairs of motile flagella, has been proposed to generate a hydrodynamic force that results in suction-based attachment via the adjacent ventral disc. We aimed to test this prevailing “hydrodynamic model” of attachment mediated by flagellar motility. We defined four distinct stages of attachment by assessing surface contacts of the trophozoite with the substrate during attachment using TIRF microscopy (TIRFM). The lateral crest of the ventral disc forms a continuous perimeter seal with the substrate, a cytological indication that trophozoites are fully attached. Using trophozoites with two types of molecularly engineered defects in flagellar beating, we determined that neither ventral flagellar beating, nor any flagellar beating, is necessary for the maintenance of attachment. Following a morpholino-based knockdown of PF16, a central pair protein, both the beating and morphology of flagella were defective, but trophozoites could still initiate proper surface contacts as seen using TIRFM and could maintain attachment in several biophysical assays. Trophozoites with impaired motility were able to attach as well as motile cells. We also generated a strain with defects in the ventral flagellar waveform by overexpressing a dominant negative form of alpha2-annexin::GFP (D122A, D275A). This dominant negative alpha2-annexin strain could initiate attachment and had only a slight decrease in the ability to withstand normal and shear forces. The time needed for attachment did increase in trophozoites with overall defective flagellar beating, however. Thus while not directly required for attachment, flagellar motility is important for positioning and orienting trophozoites prior to attachment. Drugs affecting flagellar motility may result in lower levels of attachment by indirectly limiting the number of parasites that can position the ventral disc properly against a surface and against peristaltic flow.
Zdroje
1. FlanaganPA 1992 Giardia–diagnosis, clinical course and epidemiology. A review. Epidemiol Infect 109 1 22
2. SavioliLSmithHThompsonA 2006 Giardia and Cryptosporidium join the 'Neglected Diseases Initiative. Trends Parasitol 22 203 208
3. GillinFDReinerDSMcCafferyJM 1996 Cell biology of the primitive eukaryote Giardia lamblia. Annu Rev Microbiol 50 679 705
4. AdamRD 2001 Biology of Giardia lamblia. Clin Microbiol Rev 14 447 475
5. Roxstrom-LindquistKPalmDReinerDRingqvistESvardSG 2006 Giardia immunity–an update. Trends Parasitol 22 26 31
6. HolbertonDV 1973 Mechanism of attachment of Giardia to the wall of the small intestine. Trans R Soc Trop Med Hyg 67 29 30
7. HolbertonDV 1974 Attachment of Giardia-a hydrodynamic model based on flagellar activity. J Exp Biol 60 207 221
8. DawsonSC 2010 An insider's guide to the microtubule cytoskeleton of Giardia. Cell Microbiol 12 588 598
9. DawsonSCHouseSA 2010 Life with eight flagella: flagellar assembly and division in Giardia. Curr Opin Microbiol 13 480 490
10. CampanatiLHolloschiATrosterHSpringHde SouzaW 2002 Video-microscopy observations of fast dynamic processes in the protozoon Giardia lamblia. Cell Motil Cytoskeleton 51 213 224
11. FriendDS 1966 The fine structure of Giardia muris. J Cell Biol 29 317 332
12. GingerMLPortmanNMcKeanPG 2008 Swimming with protists: perception, motility and flagellum assembly. Nat Rev Microbiol 6 838 850
13. GhoshSFrisardiMRogersRSamuelsonJ 2001 How Giardia swim and divide. Infect Immun 69 7866 7872
14. CarpenterMLCandeWZ 2009 Using morpholinos for gene knockdown in Giardia intestinalis. Eukaryot Cell 8 916 919
15. DawsonSCSagollaMSMancusoJJWoessnerDJHouseSA 2007 Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis. Eukaryot Cell 6 2354 2364
16. GaechterVSchranerEWildPHehlAB 2008 The single dynamin family protein in the primitive protozoan Giardia lamblia is essential for stage conversion and endocytic transport. Traffic 9 57 71
17. FeelyDEErlandsenSL 1981 Isolation and purification of Giardia trophozoites from rat intestine. J Parasitol 67 59 64
18. HansenWRFletcherDA 2008 Tonic Shock Induces Detachment of Giardia lamblia. PLoS Negl Trop Dis 2 e169
19. HansenWRTulyathanODawsonSCCandeWZFletcherDA 2006 Giardia lamblia attachment force is insensitive to surface treatments. Eukaryot Cell 5 781 783
20. ErlandsenSLMeyerEA 1984 Giardia and giardiasis : biology, pathogenesis, and epidemiology. New York Plenum Press xxiv 407
21. BrancheCKohlLToutiraisGBuissonJCossonJ 2006 Conserved and specific functions of axoneme components in trypanosome motility. J Cell Sci 119 3443 3455
22. SmithEFLefebvrePA 1996 PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella. J Cell Biol 132 359 370
23. SilflowCDLefebvrePA 2001 Assembly and motility of eukaryotic cilia and flagella. Lessons from Chlamydomonas reinhardtii. Plant Physiol 127 1500 1507
24. SapiroRKostetskiiIOlds-ClarkePGertonGLRadiceGL 2002 Male infertility, impaired sperm motility, and hydrocephalus in mice deficient in sperm-associated antigen 6. Mol Cell Biol 22 6298 6305
25. RalstonKSLernerAGDienerDRHillKL 2006 Flagellar motility contributes to cytokinesis in Trypanosoma brucei and is modulated by an evolutionarily conserved dynein regulatory system. Eukaryot Cell 5 696 711
26. GourguechonSCandeZW 2011 Rapid integration and tagging of genes in Giardia intestinalis. Eukaryot Cell 10 142 5
27. DutcherSKHuangBLuckDJ 1984 Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii. J Cell Biol 98 229 236
28. SagollaMSDawsonSCMancusoJJCandeWZ 2006 Three-dimensional analysis of mitosis and cytokinesis in the binucleate parasite Giardia intestinalis. J Cell Sci 119 4889 4900
29. RescherUGerkeV 2004 Annexins–unique membrane binding proteins with diverse functions. J Cell Sci 117 2631 2639
30. WeilandMEMcArthurAGMorrisonHGSoginMLSvardSG 2005 Annexin-like alpha giardins: a new cytoskeletal gene family in Giardia lamblia. Int J Parasitol 35 617 626
31. GerkeVMossSE 2002 Annexins: from structure to function. Physiol Rev 82 331 371
32. IngePMEdsonCMFarthingMJ 1988 Attachment of Giardia lamblia to rat intestinal epithelial cells. Gut 29 795 801
33. MagneDFavennecLChochillonCGorenflotAMeilletD 1991 Role of cytoskeleton and surface lectins in Giardia duodenalis attachment to Caco2 cells. J Parasitol Res 77 659 662
34. NashTEGillinFDSmithPD 1983 Excretory-secretory products of Giardia lamblia. J Immunol 131 2004 2010
35. Ortega-BarriaEWardHDKeuschGTPereiraMEA 1994 Growth Inhibition of the Intestinal Parasite Giardia lamblia by a Dietary Lectin Is Associated with Arrest of the Cell Cycle. J Clin Invest 94 2283 2288
36. SousaMCConcalvesACBairosAVPoiares-Da-SilvaJ 2001 Adherence of Giardia lamblia trophozoites to Int-407 human intestinal cells. Clin Diagn Lab Immunol 8 258 265
37. ElmendorfHGDawsonSCMcCafferyJM 2003 The cytoskeleton of Giardia lamblia. Int J Parasitol 33 3 28
38. HolbertonDV 1973 Fine structure of the ventral disk apparatus and the mechanism of attachment in the flagellate Giardia muris. J Cell Sci 13 11 41
39. PivaBBenchimolM 2004 The median body of Giardia lamblia: an ultrastructural study. Biol Cell 96 735 746
40. CorreaGBenchimolM 2006 Giardia lamblia behavior under cytochalasins treatment. Parasitol Res 98 250 256
41. MarianteRMVanciniRGMeloALBenchimolM 2005 Giardia lamblia: evaluation of the in vitro effects of nocodazole and colchicine on trophozoites. Exp Parasitol 110 62 72
42. BenchimolM 2004 Participation of the adhesive disc during karyokinesis in Giardia lamblia. Biol Cell 96 291 301
43. KattenbachWMDiniz JuniorJABenchimolMde SouzaW 1996 A deep-etch study of the cytoskeleton of Giardia duodenalis. Biol Cell 86 161 166
44. NohynkovaETumovaPKuldaJ 2006 Cell Division of Giardia intestinalis: Flagellar Developmental Cycle Involves Transformation and Exchange of Flagella between Mastigonts of a Diplomonad Cell. Eukaryot Cell 5 753 761
45. TumovaPKuldaJNohynkovaE 2007 Cell division of Giardia intestinalis: assembly and disassembly of the adhesive disc, and the cytokinesis. Cell Motil Cytoskeleton 64 288 298
46. GillinFDReinerDS 1982 Attachment of the flagellate Giardia lamblia: role of reducing agents, serum, temperature, and ionic composition. Mol Cell Biol 2 369 377
47. FeelyDEErlandsenSL 1982 Effect of cytochalasin-B, low Ca++ concentration, iodoacetic acid, and quinacrine-HCl on the attachment of Giardia trophozoites in vitro. J Parasitol 68 869 873
48. PerezPFMinnaardJRouvetMKnabenhansCBrassartD 2001 Inhibition of Giardia intestinalis by extracellular factors from Lactobacilli: an in vitro study. Appl Environ Microbiol 67 5037 5042
49. NarcisiEMPaulinJJFechheimerM 1994 Presence and localization of vinculin in Giardia. J Parasitol 80 468 473
50. FeelyDEErlandsenSL 1985 Morphology of Giardia agilis: observation by scanning electron microscopy and interference reflexion microscopy. J Protozool 32 691 693
51. PathuriPNguyenETSvardSGLueckeH 2007 Apo and calcium-bound crystal structures of Alpha-11 giardin, an unusual annexin from Giardia lamblia. J Mol Biol 368 493 508
52. WemmerKAMarshallWF 2007 Flagellar length control in chlamydomonas–paradigm for organelle size regulation. Int Rev Cytol 260 175 212
53. BabiychukEBDraegerA 2000 Annexins in cell membrane dynamics. Ca(2+)-regulated association of lipid microdomains. J Cell Biol 150 1113 1124
54. VahrmannASaricMScholzeHKoebschI 2008 alpha14-Giardin (annexin E1) is associated with tubulin in trophozoites of Giardia lamblia and forms local slubs in the flagella. Parasitol Res 102 321 326
55. GilchristCAPetriWA 1999 Virulence factors of Entamoeba histolytica. Curr Opin Microbiol 2 433 437
56. KeisterDB 1983 Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg 77 487 488
57. SunCHTaiJH 2000 Development of a tetracycline controlled gene expression system in the parasitic protozoan Giardia lamblia. Mol Biochem Parasitol 105 51 60
58. Davis-HaymanSRNashTE 2002 Genetic manipulation of Giardia lamblia. Mol Biochem Parasitol 122 1 7
59. LivakKJSchmittgenTD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 402 408
60. AxelrodD 2001 Total internal reflection fluorescence microscopy in cell biology. Traffic 2 764 774
Štítky
Hygiena a epidemiológia Infekčné lekárstvo LaboratóriumČlánok vyšiel v časopise
PLOS Pathogens
2011 Číslo 8
- Očkování proti virové hemoragické horečce Ebola experimentální vakcínou rVSVDG-ZEBOV-GP
- Parazitičtí červi v terapii Crohnovy choroby a dalších zánětlivých autoimunitních onemocnění
- Koronavirus hýbe světem: Víte jak se chránit a jak postupovat v případě podezření?
Najčítanejšie v tomto čísle
- Tumor Cell Marker PVRL4 (Nectin 4) Is an Epithelial Cell Receptor for Measles Virus
- Two Group A Streptococcal Peptide Pheromones Act through Opposing Rgg Regulators to Control Biofilm Development
- Differential Contribution of PB1-F2 to the Virulence of Highly Pathogenic H5N1 Influenza A Virus in Mammalian and Avian Species
- Recruitment of the Major Vault Protein by InlK: A Strategy to Avoid Autophagy