Editor's Choice The interaction of O157 :H7 and Typhimurium flagella with host cell membranes and cytoskeletal components Open Access

Abstract

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of O157:H7 and serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of . Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of O157:H7 and . Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from O157 (H7), EPEC O127 (H6) and . Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for O157:H7 and . Typhimurium, respectively.

Funding
This study was supported by the:
  • Biotechnology and Biological Sciences Research Council (Award BBS/E/D/20002173)
    • Principle Award Recipient: David L. Gally
  • Biotechnology and Biological Sciences Research Council (Award BBS/E/D/20231761)
    • Principle Award Recipient: David L. Gally
  • Biotechnology and Biological Sciences Research Council (Award BB/I011625/1)
    • Principle Award Recipient: David L. Gally
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000959
2020-09-04
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/166/10/947.html?itemId=/content/journal/micro/10.1099/mic.0.000959&mimeType=html&fmt=ahah

References

  1. Chaban B, Hughes HV, Beeby M. The flagellum in bacterial pathogens: for motility and a whole lot more. Semin Cell Dev Biol 2015; 46:91–103 [View Article][PubMed]
    [Google Scholar]
  2. Berg HC, Anderson RA. Bacteria swim by rotating their flagellar filaments. Nature 1973; 245:380–382 [View Article][PubMed]
    [Google Scholar]
  3. Chevance FFV, Hughes KT. Coordinating assembly of a bacterial macromolecular machine. Nat Rev Microbiol 2008; 6:455–465 [View Article][PubMed]
    [Google Scholar]
  4. Berg HC. The rotary motor of bacterial flagella. Annu Rev Biochem 2003; 72:19–54 [View Article][PubMed]
    [Google Scholar]
  5. Büttner D. Protein export according to schedule: architecture, assembly, and regulation of type III secretion systems from plant- and animal-pathogenic bacteria. Microbiol Mol Biol Rev 2012; 76:262–310 [View Article][PubMed]
    [Google Scholar]
  6. Diepold A, Armitage JP. Type III secretion systems: the bacterial flagellum and the injectisome. Philos Trans R Soc Lond B Biol Sci 2015; 370:20150020 [View Article][PubMed]
    [Google Scholar]
  7. Vonderviszt F, Uedaira H, Kidokoro S, Namba K. Structural organization of flagellin. J Mol Biol 1990; 214:97–104 [View Article][PubMed]
    [Google Scholar]
  8. Zieg J, Silverman M, Hilmen M, Simon M. Recombinational switch for gene expression. Science 1977; 196:170–172 [View Article][PubMed]
    [Google Scholar]
  9. Yonekura K, Maki-Yonekura S, Namba K. Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature 2003; 424:643–650 [View Article][PubMed]
    [Google Scholar]
  10. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001; 410:1099–1103 [View Article][PubMed]
    [Google Scholar]
  11. Zhao Y, Yang J, Shi J, Gong Y-N, Lu Q et al. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 2011; 477:596–600 [View Article][PubMed]
    [Google Scholar]
  12. Miao EA, Andersen-Nissen E, Warren SE, Aderem A. TLR5 and Ipaf: dual sensors of bacterial flagellin in the innate immune system. Semin Immunopathol 2007; 29:275–288 [View Article][PubMed]
    [Google Scholar]
  13. Yoon S-il, Kurnasov O, Natarajan V, Hong M, Gudkov AV et al. Structural basis of TLR5-flagellin recognition and signaling. Science 2012; 335:859–864 [View Article][PubMed]
    [Google Scholar]
  14. Belas R. Biofilms, flagella, and mechanosensing of surfaces by bacteria. Trends Microbiol 2014; 22:517–527 [View Article][PubMed]
    [Google Scholar]
  15. Song F, Brasch ME, Wang H, Henderson JH, Sauer K et al. How bacteria respond to material stiffness during attachment: a role of Escherichia coli flagellar motility. ACS Appl Mater Interfaces 2017; 9:22176–22184 [View Article][PubMed]
    [Google Scholar]
  16. Floyd M, Winn M, Cullen C, Sil P, Chassaing B et al. Swimming motility mediates the formation of neutrophil extracellular traps induced by flagellated Pseudomonas aeruginosa . PLoS Pathog 2016; 12:e1005987 [View Article][PubMed]
    [Google Scholar]
  17. Horstmann JA, Zschieschang E, Truschel T, de Diego J, Lunelli M et al. Flagellin phase-dependent swimming on epithelial cell surfaces contributes to productive Salmonella gut colonisation. Cell Microbiol 2017; 19:e12739 [View Article][PubMed]
    [Google Scholar]
  18. Pallen MJ, Matzke NJ. From the origin of species to the origin of bacterial flagella. Nat Rev Microbiol 2006; 4:784–790 [View Article][PubMed]
    [Google Scholar]
  19. Knutton S, Baldwin T, Williams PH, McNeish AS. Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli . Infect Immun 1989; 57:1290–1298 [View Article][PubMed]
    [Google Scholar]
  20. Goosney DL, Celli J, Kenny B, Finlay BB. Enteropathogenic Escherichia coli inhibits phagocytosis. Infect Immun 1999; 67:490–495 [View Article][PubMed]
    [Google Scholar]
  21. Hayward RD, Leong JM, Koronakis V, Campellone KG. Exploiting pathogenic Escherichia coli to model transmembrane receptor signalling. Nat Rev Microbiol 2006; 4:358–370 [View Article][PubMed]
    [Google Scholar]
  22. Hume PJ, Singh V, Davidson AC, Koronakis V. Swiss army pathogen: The Salmonella entry toolkit. Front Cell Infect Microbiol 2017; 7:LK [View Article][PubMed]
    [Google Scholar]
  23. De Souza Santos M, Orth K. The role of the Type III secretion system in the intracellular lifestyle of enteric pathogens. Microbiol Spectr 2019; 7: [View Article][PubMed]
    [Google Scholar]
  24. Mahajan A, Currie CG, Mackie S, Tree J, McAteer S et al. An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157:H7 with bovine intestinal epithelium. Cell Microbiol 2009; 11:121–137 [View Article][PubMed]
    [Google Scholar]
  25. Dziva F, van Diemen PM, Stevens MP, Smith AJ, Wallis TS. Identification of Escherichia coli O157:H7 genes influencing colonization of the bovine gastrointestinal tract using signature-tagged mutagenesis. Microbiology 2004; 150:3631–3645 [View Article][PubMed]
    [Google Scholar]
  26. Naylor SW, Gally DL, Low JC. Enterohaemorrhagic E. coli in veterinary medicine. Int J Med Microbiol 2005; 295:419–441 [View Article][PubMed]
    [Google Scholar]
  27. Erdem AL, Avelino F, Xicohtencatl-Cortes J, Girón JA. Host protein binding and adhesive properties of H6 and H7 flagella of attaching and effacing Escherichia coli . J Bacteriol 2007; 189:7426–7435 [View Article][PubMed]
    [Google Scholar]
  28. Rossez Y, Holmes A, Wolfson EB, Gally DL, Mahajan A et al. Flagella interact with ionic plant lipids to mediate adherence of pathogenic Escherichia coli to fresh produce plants. Environ Microbiol 2014; 16:2181–2195 [View Article][PubMed]
    [Google Scholar]
  29. Velge P, Wiedemann A, Rosselin M, Abed N, Boumart Z et al. Multiplicity of Salmonella entry mechanisms, a new paradigm for Salmonella pathogenesis. Microbiologyopen 2012; 1:243–258 [View Article][PubMed]
    [Google Scholar]
  30. Olsen JE, Hoegh-Andersen KH, Casadesús J, Rosenkranzt J, Chadfield MS et al. The role of flagella and chemotaxis genes in host pathogen interaction of the host adapted Salmonella enterica serovar Dublin compared to the broad host range serovar S. typhimurium. BMC Microbiol 2013; 13:67 [View Article][PubMed]
    [Google Scholar]
  31. Agbor TA, McCormick BA. Salmonella effectors: important players modulating host cell function during infection. Cell Microbiol 2011; 13:1858–1869 [View Article][PubMed]
    [Google Scholar]
  32. Rossez Y, Wolfson EB, Holmes A, Gally DL, Holden NJ. Bacterial flagella: twist and stick, or dodge across the kingdoms. PLoS Pathog 2015; 11:e1004483 [View Article][PubMed]
    [Google Scholar]
  33. Blomfield IC, Vaughn V, Rest RF, Eisenstein BI. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol Microbiol 1991; 5:1447–1457 [View Article][PubMed]
    [Google Scholar]
  34. Montie TC, Stover GB. Isolation and characterization of flagellar preparations from Pseudomonas species. J Clin Microbiol 1983; 18:452–456 [View Article][PubMed]
    [Google Scholar]
  35. Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA et al. Toll-Like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat Immunol 2003; 4:1247–1253 [View Article][PubMed]
    [Google Scholar]
  36. Schierack P, Nordhoff M, Pollmann M, Weyrauch KD, Amasheh S et al. Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine. Histochem Cell Biol 2006; 125:293–305 [View Article][PubMed]
    [Google Scholar]
  37. Mahajan A, Naylor S, Mills AD, Low JC, Mackellar A et al. Phenotypic and functional characterisation of follicle-associated epithelium of rectal lymphoid tissue. Cell Tissue Res 2005; 321:365–374 [View Article][PubMed]
    [Google Scholar]
  38. Wright CS. Structural comparison of the two distinct sugar binding sites in wheat germ agglutinin isolectin II. J Mol Biol 1984; 178:91–104 [View Article][PubMed]
    [Google Scholar]
  39. Tian L, Jeffries O, McClafferty H, Molyvdas A, Rowe ICM et al. Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels. Proc Natl Acad Sci U S A 2008; 105:21006–21011 [View Article][PubMed]
    [Google Scholar]
  40. Olmos Y, Hodgson L, Mantell J, Verkade P, Carlton JG. ESCRT-III controls nuclear envelope reformation. Nature 2015; 522:236–239 [View Article][PubMed]
    [Google Scholar]
  41. Sitthidet C, Korbsrisate S, Layton AN, Field TR, Stevens MP et al. Identification of motifs of Burkholderia pseudomallei BimA required for intracellular motility, actin binding, and actin polymerization. J Bacteriol 2011; 193:1901–1910 [View Article][PubMed]
    [Google Scholar]
  42. Van Troys M, Huyck L, Leyman S, Dhaese S, Vandekerkhove J et al. Ins and outs of ADF/cofilin activity and regulation. Eur J Cell Biol 2008; 87:649–667 [View Article][PubMed]
    [Google Scholar]
  43. Chattopadhyay S, Moldovan R, Yeung C, XL W. Swimming efficiency of bacterium. Proc Natl Acad Sci USA 2006; 103:13712–13717
    [Google Scholar]
  44. Magariyama Y, Sugiyama S, Kudo S. Bacterial swimming speed and rotation rate of bundled flagella. FEMS Microbiol Lett 2001; 199:125–129 [View Article][PubMed]
    [Google Scholar]
  45. Parish CR, Wistar R, Ada GL. Cleavage of bacterial flagellin with cyanogen bromide. antigenic properties of the protein fragments. Biochem J 1969; 113:501–506 [View Article][PubMed]
    [Google Scholar]
  46. Carlsson AE. Stimulation of actin polymerization by filament severing. Biophys J 2006; 90:413–422 [View Article][PubMed]
    [Google Scholar]
  47. Crawford RW, Reeve KE, Gunn JS. Flagellated but not hyperfimbriated Salmonella enterica serovar Typhimurium attaches to and forms biofilms on cholesterol-coated surfaces. J Bacteriol 2010; 192:2981–2990 [View Article][PubMed]
    [Google Scholar]
  48. Friedlander RS, Vogel N, Aizenberg J. Role of flagella in adhesion of Escherichia coli to abiotic surfaces. Langmuir 2015; 31:6137–6144 [View Article][PubMed]
    [Google Scholar]
  49. Qi M, Gong X, Wu B, Zhang G. Landing dynamics of swimming bacteria on a polymeric surface: effect of surface properties. Langmuir 2017; 33:3525–3533 [View Article][PubMed]
    [Google Scholar]
  50. Misselwitz B, Barrett N, Kreibich S, Vonaesch P, Andritschke D et al. Near surface swimming of Salmonella Typhimurium explains target-site selection and cooperative invasion. PLoS Pathog 2012; 8:e1002810 [View Article][PubMed]
    [Google Scholar]
  51. Friedlander RS, Vlamakis H, Kim P, Khan M, Kolter R et al. Bacterial flagella explore microscale hummocks and hollows to increase adhesion. Proc Natl Acad Sci U S A 2013; 110:5624–5629 [View Article][PubMed]
    [Google Scholar]
  52. Bucior I, Pielage JF, Engel JN. Pseudomonas aeruginosa pili and flagella mediate distinct binding and signaling events at the apical and basolateral surface of airway epithelium. PLoS Pathog 2012; 8:e1002616 [View Article][PubMed]
    [Google Scholar]
  53. Lillehoj EP, Kim BT, Kim KC. Identification of Pseudomonas aeruginosa flagellin as an adhesin for Muc1 mucin. Am J Physiol Lung Cell Mol Physiol 2002; 282:L751–L756 [View Article]
    [Google Scholar]
  54. Kato K, Lillehoj EP, Park YS, Umehara T, Hoffman NE et al. Membrane-Tethered MUC1 mucin is phosphorylated by epidermal growth factor receptor in airway epithelial cells and associates with TLR5 to inhibit recruitment of MyD88. J Immunol 2012; 188:2014–2022 [View Article][PubMed]
    [Google Scholar]
  55. Schmitt CK, Ikeda JS, Darnell SC, Watson PR, Bispham J et al. Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis. Infect Immun 2001; 69:5619–5625 [View Article][PubMed]
    [Google Scholar]
  56. Mellmann A, Bielaszewska M, Köck R, Friedrich AW, Fruth A et al. Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli . Emerg Infect Dis 2008; 14:1287–1290 [View Article][PubMed]
    [Google Scholar]
  57. Girón JA, Torres AG, Freer E, Kaper JB. The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol Microbiol 2002; 44:361–379 [View Article][PubMed]
    [Google Scholar]
  58. Eckhard U, Bandukwala H, Mansfield MJ, Marino G, Cheng J et al. Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella. Nat Commun 2017; 8:521 [View Article][PubMed]
    [Google Scholar]
  59. Tilney LG, Portnoy DA. Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J Cell Biol 1989; 109:1597–1608 [View Article][PubMed]
    [Google Scholar]
  60. Gouin E, Gantelet H, Egile C, Lasa I, Ohayon H et al. A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conorii . J Cell Sci 1999; 112:1697–1708[PubMed]
    [Google Scholar]
  61. Patel JC, Galán JE. Manipulation of the host actin cytoskeleton by Salmonella--all in the name of entry. Curr Opin Microbiol 2005; 8:10–15 [View Article][PubMed]
    [Google Scholar]
  62. Caron E, Crepin VF, Simpson N, Knutton S, Garmendia J et al. Subversion of actin dynamics by EPEC and EHEC. Curr Opin Microbiol 2006; 9:40–45 [View Article][PubMed]
    [Google Scholar]
  63. Sirisaengtaksin N, Odem MA, Bosserman RE, Flores EM, Krachler AM. The E. coli transcription factor GrlA is regulated by subcellular compartmentalization and activated in response to mechanical stimuli. Proc Natl Acad Sci U S A 2020; 117:9519–9528 [View Article][PubMed]
    [Google Scholar]
  64. Slater SL, Sågfors AM, Pollard DJ, Ruano-Gallego D, Frankel G. The type III secretion system of pathogenic Escherichia coli . Curr Top Microbiol Immunol 2018; 416:51–72 [View Article][PubMed]
    [Google Scholar]
  65. Horstmann JA, Lunelli M, Cazzola H, Heidemann J, Kühne C et al. Methylation of Salmonella Typhimurium flagella promotes bacterial adhesion and host cell invasion. Nat Commun 2020; 11:11 [View Article][PubMed]
    [Google Scholar]
  66. Man SM, Ekpenyong A, Tourlomousis P, Achouri S, Cammarota E et al. Actin polymerization as a key innate immune effector mechanism to control Salmonella infection. Proc Natl Acad Sci U S A 2014; 111:17588–17593 [View Article][PubMed]
    [Google Scholar]
  67. Campellone KG, Roe AJ, Løbner-Olesen A, Murphy KC, Magoun L et al. Increased adherence and actin pedestal formation by dam-deficient enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol 2007; 63:1468–1481 [View Article][PubMed]
    [Google Scholar]
  68. Iguchi A, Thomson NR, Ogura Y, Saunders D, Ooka T et al. Complete genome sequence and comparative genome analysis of enteropathogenic Escherichia coli O127:H6 strain E2348/69. J Bacteriol 2009; 191:347–354 [View Article][PubMed]
    [Google Scholar]
  69. Blattner FR, Plunkett G, Bloch CA, Perna NT, Burland V et al. The complete genome sequence of Escherichia coli K-12. Science 1997; 277:1453–1462 [View Article][PubMed]
    [Google Scholar]
  70. Flockhart AF, Tree JJ, Xu X, Karpiyevich M, McAteer SP et al. Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion. Mol Microbiol 2012; 83:208–223 [View Article][PubMed]
    [Google Scholar]
  71. Arques JL, Hautefort I, Ivory K, Bertelli E, Regoli M et al. Salmonella induces flagellin- and MyD88-dependent migration of bacteria-capturing dendritic cells into the gut lumen. Gastroenterology 2009; 137:579–587 [View Article][PubMed]
    [Google Scholar]
  72. Richardson EJ, Limaye B, Inamdar H, Datta A, Manjari KS et al. Genome sequences of Salmonella enterica serovar typhimurium, choleraesuis, Dublin and gallinarum strains of well- defined virulence in food-producing animals. J Bacteriol 2011; 193:3162–3163 [View Article][PubMed]
    [Google Scholar]
  73. Morgan E, Campbell JD, Rowe SC, Bispham J, Stevens MP et al. Identification of host-specific colonization factors of Salmonella enterica serovar Typhimurium. Mol Microbiol 2004; 54:994–1010 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000959
Loading
/content/journal/micro/10.1099/mic.0.000959
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

Supplementary material 3

MOVIE

Supplementary material 4

MOVIE

Supplementary material 5

MOVIE

Most cited Most Cited RSS feed