Functional analyses of pilin-like proteins from : complementation of type IV pilus phenotypes in Free

Abstract

Accumulating evidence from a number of studies strongly suggests that proteins orthologous to those involved in type IV pili (Tfp) assembly and function are required for pathogenicity. However, the molecular mechanisms by which the components exert their influence on virulence remain poorly understood. Owing to the conservation and promiscuity of Tfp biogenesis machineries, expression of Tfp pilins in heterologous species has been used successfully to analyse organelle structure–function relationships. In this study we expressed a number of pilin genes in the Tfp-expressing pathogen lacking its endogenous pilin subunit. Two gene products, the orthologous PilA proteins from subspecies and , were capable of restoring the expression of Tfp-like appendages that were shown to be dependent upon the neisserial Tfp biogenesis machinery for surface localization. Expression of PilA pilins also partially restored competence for natural transformation in . This phenotype was not complemented by expression of the PulG and XcpT proteins, which are equivalent components of the related type II protein secretion system. Taken together, these findings provide compelling, although indirect, evidence of the potential for PilA proteins to express functional Tfp.

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2009-08-01
2024-03-28
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References

  1. Aas F., Wolfgang M., Frye S., Dunham S., Løvold C., Koomey M. 2002; Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression. Mol Microbiol 46:749–760
    [Google Scholar]
  2. Aas F., Egge-Jacobsen W., Winther-Larsen H., Løvold C., Hitchen P., Dell A., Koomey M. 2006; Neisseria gonorrhoeae type IV pili undergo multisite, hierarchical modifications with phosphoethanolamine and phosphocholine requiring an enzyme structurally related to lipopolysaccharide phosphoethanolamine transferases. J Biol Chem 281:27712–27723
    [Google Scholar]
  3. Aas F., Vik A., Vedde J., Koomey M., Egge-Jacobsen W. 2007a; Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure. Mol Microbiol 65:607–624
    [Google Scholar]
  4. Aas F., Winther-Larsen H., Wolfgang M., Frye S., Løvold C., Roos N., van Putten J., Koomey M. 2007b; Substitutions in the N-terminal alpha helical spine of Neisseria gonorrhoeae pilin affect Type IV pilus assembly, dynamics and associated functions. Mol Microbiol 63:69–85
    [Google Scholar]
  5. Alm R., Mattick J. 1996; Identification of two genes with prepilin-like leader sequences involved in type 4 fimbrial biogenesis in Pseudomonas aeruginosa . J Bacteriol 178:3809–3817
    [Google Scholar]
  6. Alm R., Bodero A., Free P., Mattick J. 1996a; Identification of a novel gene, pilZ, essential for type 4 fimbrial biogenesis in Pseudomonas aeruginosa . J Bacteriol 178:46–53
    [Google Scholar]
  7. Alm R., Hallinan J., Watson A., Mattick J. 1996b; Fimbrial biogenesis genes of Pseudomonas aeruginosa: pilW and pilX increase the similarity of type 4 fimbriae to the GSP protein-secretion systems and pilY1 encodes a gonococcal PilC homologue. Mol Microbiol 22:161–173
    [Google Scholar]
  8. Bradley D., Pitt T. 1974; Pilus-dependence of four Pseudomonas aeruginosa bacteriophages with non-contractile tails. J Gen Virol 24:1–15
    [Google Scholar]
  9. Carbonnelle E., Helaine S., Nassif X., Pelicic V. 2006; A systematic genetic analysis in Neisseria meningitidis defines the Pil proteins required for assembly, functionality, stabilization and export of type IV pili. Mol Microbiol 61:1510–1522
    [Google Scholar]
  10. Chakraborty S., Monfett M., Maier T., Benach J., Frank D., Thanassi D. 2008; Type IV pili in Francisella tularensis: roles of pilF and pilT in fiber assembly, host cell adherence, and virulence. Infect Immun 76:2852–2861
    [Google Scholar]
  11. d'Enfert C., Pugsley A. 1987; A gene fusion approach to the study of pullulanase export and secretion in Escherichia coli . Mol Microbiol 1:159–168
    [Google Scholar]
  12. Dennis D., Inglesby T., Henderson D., Bartlett J. G., Ascher M. S., Eitzen E., Fine A. D., Friedlander A. M., Hauer J. other authors 2001; Tularemia as a biological weapon: medical and public health management. JAMA 285:2763–2773
    [Google Scholar]
  13. Durand E., Bernadac A., Ball G., Lazdunski A., Sturgis J., Filloux A. 2003; Type II protein secretion in Pseudomonas aeruginosa: the pseudopilus is a multifibrillar and adhesive structure. J Bacteriol 185:2749–2758
    [Google Scholar]
  14. Elleman T., Hoyne P., Stewart D., McKern N., Peterson J. 1986; Expression of pili from Bacteroides nodosus in Pseudomonas aeruginosa . J Bacteriol 168:574–580
    [Google Scholar]
  15. Forsberg A., Guina T. 2007; Type II secretion and type IV pili of Francisella . Ann N Y Acad Sci 1105187–201
    [Google Scholar]
  16. Forslund A., Kuoppa K., Svensson K., Salomonsson E., Johansson A., Byström M., Oyston P. C., Michell S. L., Titball R. W. other authors 2006; Direct repeat-mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis . Mol Microbiol 59:1818–1830
    [Google Scholar]
  17. Freitag N., Seifert H., Koomey M. 1995; Characterization of the pilF- pilD pilus-assembly locus of Neisseria gonorrhoeae . Mol Microbiol 16:575–586
    [Google Scholar]
  18. Fullner K., Mekalanos J. 1999; Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae . Infect Immun 67:1393–1404
    [Google Scholar]
  19. Gallagher L., McKevitt M., Ramage E., Manoil C. 2008; Genetic dissection of the Francisella novicida restriction barrier. J Bacteriol 190:7830–7837
    [Google Scholar]
  20. Gil H., Benach J., Thanassi D. 2004; Presence of pili on the surface of Francisella tularensis . Infect Immun 72:3042–3047
    [Google Scholar]
  21. Golovliov I., Sjöstedt A., Mokrievich A., Pavlov V. 2003; A method for allelic replacement in Francisella tularensis . FEMS Microbiol Lett 222:273–280
    [Google Scholar]
  22. Hager A., Bolton D., Pelletier M., Brittnacher M., Gallagher L., Kaul R., Skerrett S., Miller S., Guina T. 2006; Type IV pili-mediated secretion modulates Francisella virulence. Mol Microbiol 62:227–237
    [Google Scholar]
  23. Han X., Kennan R., Davies J., Reddacliff L., Dhungyel O., Whittington R., Turnbull L., Whitchurch C., Rood J. 2008; Twitching motility is essential for virulence in Dichelobacter nodosus . J Bacteriol 190:3323–3335
    [Google Scholar]
  24. Hegge F., Hitchen P., Aas F., Kristiansen H., Løvold C., Egge-Jacobsen W., Panico M., Leong W. Y., Bull V. other authors 2004; Unique modifications with phosphocholine and phosphoethanolamine define alternate antigenic forms of Neisseria gonorrhoeae type IV pili. Proc Natl Acad Sci U S A 101:10798–10803
    [Google Scholar]
  25. Helaine S., Dyer D., Nassif X., Pelicic V., Forest K. 2007; 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili. Proc Natl Acad Sci U S A 104:15888–15893
    [Google Scholar]
  26. Hollis D., Weaver R., Steigerwalt A., Wenger J., Moss C., Brenner D. 1989; Francisella philomiragia comb. nov. (formerly Yersinia philomiragia) and Francisella tularensis biogroup novicida (formerly Francisella novicida) associated with human disease. J Clin Microbiol 27:1601–1608
    [Google Scholar]
  27. Hoyne P., Haas R., Meyer T., Davies J., Elleman T. 1992; Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa . J Bacteriol 174:7321–7327
    [Google Scholar]
  28. Källström H., Islam M., Berggren P., Jonsson A. 1998; Cell signaling by the type IV pili of pathogenic Neisseria . J Biol Chem 273:21777–21782
    [Google Scholar]
  29. Khan A., Morse S., Lillibridge S. 2000; Public-health preparedness for biological terrorism in the USA. Lancet 356:1179–1182
    [Google Scholar]
  30. Kirn T., Bose N., Taylor R. 2003; Secretion of a soluble colonization factor by the TCP type 4 pilus biogenesis pathway in Vibrio cholerae . Mol Microbiol 49:81–92
    [Google Scholar]
  31. Koomey J., Falkow S. 1987; Cloning of the recA gene of Neisseria gonorrhoeae and construction of gonococcal recA mutants. J Bacteriol 169:790–795
    [Google Scholar]
  32. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  33. Larsson P., Oyston P., Chain P., Chu M. C., Duffield M., Fuxelius H. H., Garcia E., Hälltorp G., Johansson D. other authors 2005; The complete genome sequence of Francisella tularensis, the causative agent of tularemia. Nat Genet 37:153–159
    [Google Scholar]
  34. Long C., Hayes S., van Putten J., Harvey H., Apicella M., Seifert H. 2001; Modulation of gonococcal piliation by regulatable transcription of pilE . J Bacteriol 183:1600–1609
    [Google Scholar]
  35. Lu H., Motley S., Lory S. 1997; Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion. Mol Microbiol 25:247–259
    [Google Scholar]
  36. Maier B., Koomey M., Sheetz M. 2004; A force-dependent switch reverses type IV pilus retraction. Proc Natl Acad Sci U S A 101:10961–10966
    [Google Scholar]
  37. Martin P., Watson A., McCaul T., Mattick J. 1995; Characterization of a five-gene cluster required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa . Mol Microbiol 16:497–508
    [Google Scholar]
  38. Mattick J. 2002; Type IV pili and twitching motility. Annu Rev Microbiol 56:289–314
    [Google Scholar]
  39. Mattick J., Whitchurch C., Alm R. 1996; The molecular genetics of type-4 fimbriae in Pseudomonas aeruginosa – a review. Gene 179:147–155
    [Google Scholar]
  40. Merz A., So M., Sheetz M. 2000; Pilus retraction powers bacterial twitching motility. Nature 407:98–102
    [Google Scholar]
  41. Mörner T. 1992; The ecology of tularaemia. Rev Sci Tech 11:1123–1130
    [Google Scholar]
  42. Nudleman E., Kaiser D. 2004; Pulling together with type IV pili. J Mol Microbiol Biotechnol 7:52–62
    [Google Scholar]
  43. Nudleman E., Wall D., Kaiser D. 2006; Polar assembly of the type IV pilus secretin in Myxococcus xanthus . Mol Microbiol 60:16–29
    [Google Scholar]
  44. O'Toole G., Kolter R. 1998; Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30:295–304
    [Google Scholar]
  45. Park H., Wolfgang M., Koomey M. 2002; Modification of type IV pilus-associated epithelial cell adherence and multicellular behavior by the PilU protein of Neisseria gonorrhoeae . Infect Immun 70:3891–3903
    [Google Scholar]
  46. Patel P., Marrs C., Mattick J., Ruehl W., Taylor R., Koomey M. 1991; Shared antigenicity and immunogenicity of type 4 pilins expressed by Pseudomonas aeruginosa, Moraxella bovis, Neisseria gonorrhoeae, Dichelobacter nodosus, and Vibrio cholerae . Infect Immun 59:4674–4676
    [Google Scholar]
  47. Peabody C., Chung Y., Yen M., Vidal-Ingigliardi D., Pugsley A., Saier M. J. 2003; Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. Microbiology 149:3051–3072
    [Google Scholar]
  48. Petersen J., Schriefer M. 2005; Tularemia: emergence/re-emergence. Vet Res 36:455–467
    [Google Scholar]
  49. Rohmer L., Fong C., Abmayr S., Wasnick M., Larson Freeman T. J., Radey M., Guina T., Svensson K., Hayden H. S. other authors 2007; Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains. Genome Biol 8:R102
    [Google Scholar]
  50. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  51. Sandström G., Tärnvik A., Wolf-Watz H., Löfgren S. 1984; Antigen from Francisella tularensis: nonidentity between determinants participating in cell-mediated and humoral reactions. Infect Immun 45:101–106
    [Google Scholar]
  52. Satyshur K., Worzalla G., Meyer L., Heiniger E., Aukema K., Misic A., Forest K. 2007; Crystal structures of the pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility. Structure 15:363–376
    [Google Scholar]
  53. Sauvonnet N., Gounon P., Pugsley A. 2000; PpdD type IV pilin of Escherichia coli K-12 can be assembled into pili in Pseudomonas aeruginosa . J Bacteriol 182:848–854
    [Google Scholar]
  54. Skerker J., Berg H. 2001; Direct observation of extension and retraction of type IV pili. Proc Natl Acad Sci U S A 98:6901–6904
    [Google Scholar]
  55. Strom M., Lory S. 1993; Structure-function and biogenesis of the type IV pili. Annu Rev Microbiol 47:565–596
    [Google Scholar]
  56. Strom M., Nunn D., Lory S. 1993; A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Proc Natl Acad Sci U S A 90:2404–2408
    [Google Scholar]
  57. Svensson K., Larsson P., Johansson D., Byström M., Forsman M., Johansson A. 2005; Evolution of subspecies of Francisella tularensis . J Bacteriol 187:3903–3908
    [Google Scholar]
  58. Tärnvik A. 1989; Nature of protective immunity to Francisella tularensis . Rev Infect Dis 11:440–451
    [Google Scholar]
  59. Tärnvik A., Berglund L. 2003; Tularaemia. Eur Respir J 21:361–373
    [Google Scholar]
  60. Tønjum T., Koomey M. 1997; The pilus colonization factor of pathogenic neisserial species: organelle biogenesis and structure/function relationships – a review. Gene 192:155–163
    [Google Scholar]
  61. Tønjum T., Freitag N., Namork E., Koomey M. 1995; Identification and characterization of pilG, a highly conserved pilus-assembly gene in pathogenic Neisseria . Mol Microbiol 16:451–464
    [Google Scholar]
  62. Tyeryar F., Lawton W. 1970; Factors affecting transformation of Pasteurella novicida . J Bacteriol 104:1312–1317
    [Google Scholar]
  63. Vignon G., Köhler R., Larquet E., Giroux S., Prévost M., Roux P., Pugsley A. 2003; Type IV-like pili formed by the type II secreton: specificity, composition, bundling, polar localization and surface presentation of peptides. J Bacteriol 185:3416–3428
    [Google Scholar]
  64. Vik A., Aas F., Anonsen J., Bilsborough S., Schneider A., Egge-Jacobsen W., Koomey M. 2009; Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae . Proc Natl Acad Sci U S A 106:4447–4452
    [Google Scholar]
  65. Waldor M., Mekalanos J. 1996; Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272:1910–1914
    [Google Scholar]
  66. Whipp M., Davis J., Lum G., de Boer J., Zhou Y., Bearden S., Petersen J., Chu M., Hogg G. 2003; Characterization of a novicida-like subspecies of Francisella tularensis isolated in Australia. J Med Microbiol 52:839–842
    [Google Scholar]
  67. Whitchurch C., Hobbs M., Livingston S., Krishnapillai V., Mattick J. 1991; Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. Gene 101:33–44
    [Google Scholar]
  68. Winther-Larsen H., Hegge F., Wolfgang M., Hayes S., van Putten J., Koomey M. 2001; Neisseria gonorrhoeae PilV, a type IV pilus-associated protein essential to human epithelial cell adherence. Proc Natl Acad Sci U S A 98:15276–15281
    [Google Scholar]
  69. Winther-Larsen H., Wolfgang M., Dunham S., van Putten J., Dorward D., Løvold C., Aas F., Koomey M. 2005; A conserved set of pilin-like molecules controls type IV pilus dynamics and organelle-associated functions in Neisseria gonorrhoeae . Mol Microbiol 56:903–917
    [Google Scholar]
  70. Winther-Larsen H., Wolfgang M., van Putten J., Roos N., Aas F., Egge-Jacobsen W., Maier B., Koomey M. 2007; Pseudomonas aeruginosa Type IV pilus expression in Neisseria gonorrhoeae: effects of pilin subunit composition on function and organelle dynamics. J Bacteriol 189:6676–6685
    [Google Scholar]
  71. Wolfgang M., Lauer P., Park H., Brossay L., Hébert J., Koomey M. 1998a; PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae . Mol Microbiol 29:321–330
    [Google Scholar]
  72. Wolfgang M., Park H., Hayes S., van Putten J., Koomey M. 1998b; Suppression of an absolute defect in type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae . Proc Natl Acad Sci U S A 95:14973–14978
    [Google Scholar]
  73. Wolfgang M., van Putten J., Hayes S., Koomey M. 1999; The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation. Mol Microbiol 31:1345–1357
    [Google Scholar]
  74. Wolfgang M., van Putten J., Hayes S., Dorward D., Koomey M. 2000; Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili. EMBO J 19:6408–6418
    [Google Scholar]
  75. Zogaj X., Chakraborty S., Liu J., Thanassi D., Klose K. 2008; Characterization of the Francisella tularensis subsp. novicida type IV pilus. Microbiology 154:2139–2150
    [Google Scholar]
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