Shearing and Enrichment of Extracellular Type IV Pili

  • Alba Katiria Gonzalez Rivera
  • Katrina T. ForestEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)


Pili are widespread among bacteria. Type IVa pili (T4aP) are associated with a variety of bacterial functions, including adhesion, motility, natural transformation, biofilm formation, and force-dependent signaling. In pathogenic bacteria, T4aP play a crucial role during infection and have been the subject of hundreds of studies. Methods for the isolation and purification of T4aP were first described in the 1970s. Purified pili have been used for studies of filament protein content, morphology, immunogenicity, post-translational modifications, and X-ray crystallography. We detail a tried-and-true method of isolating large amounts of native T4aP from bacterial surfaces. The method requires supplies and equipment that are available in most microbiology labs.

Key words

T4P Fimbriae Pili Filament Shear Isolation 



We are grateful to Dr. Nicole Koropatkin for purifying grams of pili and optimizing this protocol in the process, and to Dr. Lisa Craig for many years of collegial interactions and helpful suggestions for this chapter.


  1. 1.
    Hansen JK, Forest KT (2006) Type IV pilin structures: insights on shared architecture, fiber assembly, receptor binding and type II secretion. J Mol Microbiol Biotechnol 11:192–207CrossRefPubMedGoogle Scholar
  2. 2.
    MacRae TH, Dobson WJ, McCurdy HD (1977) Fimbriation in gliding bacteria. Can J Microbiol 23:1096–1108CrossRefPubMedGoogle Scholar
  3. 3.
    Henrichsen J, Blom J (1975) Examination of fimbriation of some gram-negative rods with and without twitching and gliding motility. Acta Pathol Microbiol Scand B 83:161–170PubMedGoogle Scholar
  4. 4.
    Swanson J (1973) Studies on gonococcus infection. IV. Pili: their role in attachment of gonococci to tissue culture cells. J Exp Med 137:571–589CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Saarimaa C, Peltola M, Raulio M, Neu TR, Salkinoja-Salonen MS, Neubauer P (2006) Characterization of adhesion threads of Deinococcus geothermalis as type IV pili. J Bacteriol 188:7016–7021CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Imam S, Chen Z, Roos DS, Pohlschroder M (2011) Identification of surprisingly diverse type IV pili, across a broad range of gram-positive bacteria. PLoS One 6:e28919CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Melville S, Craig L (2013) Type IV pili in Gram-positive bacteria. Microbiol Mol Biol Rev 77:323–341CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Berry JL, Pelicic V (2015) Exceptionally widespread nanomachines composed of type IV pilins: the prokaryotic Swiss Army knives. FEMS Microbiol Rev 39:134–154CrossRefPubMedGoogle Scholar
  9. 9.
    Pelicic V (2008) Type IV pili: e pluribus unum? Mol Microbiol 68:827–837CrossRefPubMedGoogle Scholar
  10. 10.
    Brinton CC, Bryan J, Dillon J-A, Guerina N, Jen Jacobson L, Labik A, Lee S, McMichael J, Polen S, Rogers K, ACC T, SCM T (1978) Uses of pili in Gonorrhea control: role of bacterial pili in disease, purification and properties of Gonococcal pilus vaccine for Gonorrhea. In: Brooks GF (ed) Immunobiology of Neisseria gonorrhoeae: proceedings of a conference held in San Francisco, CA. American Society for Microbiology, Washington, DCGoogle Scholar
  11. 11.
    Li J, Lim MS, Li S, Brock M, Pique ME, Woods VL Jr, Craig L (2008) Vibrio cholerae toxin-coregulated pilus structure analyzed by hydrogen/deuterium exchange mass spectrometry. Structure 16:137–148CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Forest KT, Dunham SA, Koomey M, Tainer JA (1999) Crystallographic structure reveals phosphorylated pilin from Neisseria: phosphoserine sites modify type IV pilus surface chemistry and fibre morphology. Mol Microbiol 31:743–752CrossRefPubMedGoogle Scholar
  13. 13.
    Parge HE, Bernstein SL, Deal CD, McRee DE, Christensen D, Capozza MA, Kays BW, Fieser TM, Draper D, So M (1990) Biochemical purification and crystallographic characterization of the fiber-forming protein pilin from Neisseria gonorrhoeae. J Biol Chem 265:2278–2285PubMedGoogle Scholar
  14. 14.
    Parge HE, Forest KT, Hickey MJ, Christensen DA, Getzoff ED, Tainer JA (1995) Structure of the fibre-forming protein pilin at 2.6 Å resolution. Nature 378:32–38CrossRefPubMedGoogle Scholar
  15. 15.
    Craig L, Taylor RK, Pique ME, Adair BD, Arvai AS, Singh M, Lloyd SJ, Shin DS, Getzoff ED, Yeager M, Forest KT, Tainer JA (2003) Type IV pilin structure and assembly: X-ray and EM analyses of Vibrio cholerae toxin-coregulated pilus and Pseudomonas aeruginosa PAK pilin. Mol Cell 11:1139–1150CrossRefPubMedGoogle Scholar
  16. 16.
    Schagger H (2006) Tricine-SDS-PAGE. Nat Protoc 1:16–22CrossRefPubMedGoogle Scholar
  17. 17.
    Bradley DE (1974) The adsorption of Pseudomonas aeruginosa pilus-dependent bacteriophages to a host mutant with nonretractile pili. Virology 58:149–163CrossRefPubMedGoogle Scholar
  18. 18.
    Whitchurch CB, Hobbs M, Livingston SP, Krishnapillai V, Mattick JS (1991) Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. Gene 101:33–44CrossRefPubMedGoogle Scholar
  19. 19.
    Bradley DE (1980) A function of Pseudomonas aeruginosa PAO polar pili: twitching motility. Can J Microbiol 26:146–154CrossRefPubMedGoogle Scholar
  20. 20.
    Frost LS, Paranchych W (1977) Composition and molecular weight of pili purified from Pseudomonas aeruginosa K. J Bacteriol 131:259–269PubMedPubMedCentralGoogle Scholar
  21. 21.
    Han X, Kennan RM, Davies JK, Reddacliff LA, Dhungyel OP, Whittington RJ, Turnbull L, Whitchurch CB, Rood JI (2008) Twitching motility is essential for virulence in Dichelobacter nodosus. J Bacteriol 190:3323–3335CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Meng Y, Li Y, Galvani CD, Hao G, Turner JN, Burr TJ, Hoch HC (2005) Upstream migration of Xylella fastidiosa via pilus-driven twitching motility. J Bacteriol 187:5560–5567CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, de Castro E, Duvaud S, Flegel V, Fortier A, Gasteiger E, Grosdidier A, Hernandez C, Ioannidis V, Kuznetsov D, Liechti R, Moretti S, Mostaguir K, Redaschi N, Rossier G, Xenarios I, Stockinger H (2012) ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res 40:W597–W603CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Alba Katiria Gonzalez Rivera
    • 1
  • Katrina T. Forest
    • 1
    Email author
  1. 1.Department of Bacteriology and Biophysics ProgramUniversity of Wisconsin-MadisonMadisonUSA

Personalised recommendations