Letter | Published:

Enterotoxigenic Escherichia coli EtpA mediates adhesion between flagella and host cells

Nature volume 457, pages 594598 (29 January 2009) | Download Citation



Adhesion to epithelial cells1 and flagella-mediated motility are critical virulence traits for many Gram-negative pathogens, including enterotoxigenic Escherichia coli (ETEC)2, a major cause of diarrhoea in travellers and children in developing countries3,4. Many flagellated pathogens export putative adhesins belonging to the two-partner secretion (TPS) family5. However, the actual function of these adhesins remains largely undefined. Here we demonstrate that EtpA, a TPS exoprotein adhesin of enterotoxigenic E. coli6, mimics and interacts with highly conserved regions of flagellin, the major subunit of flagella, and that these interactions are critical for adherence and intestinal colonization. Although conserved regions of flagellin are mostly buried in the flagellar shaft7, our results suggest that they are at least transiently exposed at the tips of flagella where they capture EtpA adhesin molecules for presentation to eukaryotic receptors. Similarity of EtpA to molecules encoded by other motile pathogens suggests a potential common pattern for bacterial adhesion, whereas participation of conserved regions of flagellin in adherence has implications for development of vaccines for Gram-negative pathogens.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    , & Adherence of diarrheagenic Escherichia coli strains to epithelial cells. Infect. Immun. 73, 18–29 (2005)

  2. 2.

    , & Directed delivery of heat-labile enterotoxin by enterotoxigenic Escherichia coli. Cell. Microbiol. 8, 1516–1527 (2006)

  3. 3.

    World Health Organization. Future directions for research on enterotoxigenic Escherichia coli vaccines for developing countries. Wkly Epidemiol. Rec. 81, 97–104 (2006)

  4. 4.

    et al. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin. Microbiol. Rev. 18, 465–483 (2005)

  5. 5.

    , & Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins. Mol. Microbiol. 40, 306–313 (2001)

  6. 6.

    et al. Identification of a two-partner secretion locus of enterotoxigenic Escherichia coli. Infect. Immun. 74, 2245–2258 (2006)

  7. 7.

    , & Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature 424, 643–650 (2003)

  8. 8.

    et al. Plasmid-controlled colonization factor associated with virulence in Escherichia coli enterotoxigenic for humans. Infect. Immun. 12, 656–667 (1975)

  9. 9.

    & Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 4, 444–452 (1996)

  10. 10.

    & Vaccines against enterotoxigenic Escherichia coli. Expert Rev. Vaccines 7, 795–804 (2008)

  11. 11.

    et al. The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol. Microbiol. 44, 361–379 (2002)

  12. 12.

    Occurrence, distribution, and associations of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli. Clin. Microbiol. Rev. 10, 569–584 (1997)

  13. 13.

    et al. The bacterial flagellar cap as the rotary promoter of flagellin self-assembly. Science 290, 2148–2152 (2000)

  14. 14.

    in Protein–Protein Interactions: A Molecular Cloning Manual 2nd edn, Vol. 1 (eds Golemis, E. & Adams, P. D.) Ch. 3 37–54 (Cold Spring Harbor Laboratory Press, 2005)

  15. 15.

    et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997)

  16. 16.

    et al. Flagellar proteins and type III-exported virulence factors are the predominant proteins secreted into the culture media of Salmonella typhimurium. Mol. Microbiol. 34, 767–779 (1999)

  17. 17.

    & Role of the Hrp pilus in type III protein secretion in Pseudomonas syringae. Science 294, 2556–2558 (2001)

  18. 18.

    , & Domain movements of HAP2 in the cap-filament complex formation and growth process of the bacterial flagellum. Proc. Natl Acad. Sci. USA 100, 15528–15533 (2003)

  19. 19.

    & Linker insertion mutagenesis as probe of structure–function relationships. Methods Enzymol. 208, 586–603 (1991)

  20. 20.

    et al. The EtpA exoprotein of enterotoxigenic Escherichia coli promotes intestinal colonization and is a protective antigen in an experimental model of murine infection. Infect. Immun. 76, 2106–2112 (2008)

  21. 21.

    & Roles for motility in bacterial–host interactions. Mol. Microbiol. 24, 1109–1117 (1997)

  22. 22.

    Vaccines: correlates of vaccine-induced immunity. Clin. Infect. Dis. 47, 401–409 (2008)

  23. 23.

    , & The three-dimensional structure of CFA/I adhesion pili: traveler’s diarrhea bacteria hang on by a spring. J. Mol. Biol. 376, 614–620 (2008)

  24. 24.

    et al. Surface anchoring of a bacterial adhesin secreted by the two-partner secretion pathway. Mol. Microbiol. 61, 470–483 (2006)

  25. 25.

    et al. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nature Immunol. 4, 1247–1253 (2003)

  26. 26.

    , & Importance of heat-labile enterotoxin in colonization of the adult mouse small intestine by human enterotoxigenic Escherichia coli strains. Infect. Immun. 74, 869–875 (2006)

  27. 27.

    et al. High-throughput proteomics processing of proteins in polyacrylamide in a multiwell format. J. Proteome Res. 6, 1603–1608 (2007)

  28. 28.

    & ProFound: an expert system for protein identification using mass spectrometric peptide mapping information. Anal. Chem. 72, 2482–2489 (2000)

  29. 29.

    et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410, 1099–1103 (2001)

Download references


We thank B. Westerlund-Wikström for supplying anti-flagellar antisera, K. Troughton of the Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, for her assistance with transmission electron microscopy, and L. Boykins of the Integrated Microscopy Center at the University of Memphis for her assistance with scanning electron microscopy. We thank G. Byrne, H. Courtney, J. Dale, S. Dagogo-Jack and T. Strom for reading the manuscript. This research was supported by grants from the National Institutes of Health (National Center for Research Resources) RR16190-05, the Department of Veterans Affairs and funds from the University of Tennessee Microbial Pathogenesis Research Center.

Author Contributions K.R. performed adherence assays, transmission electron and confocal microscopy, mouse colonization studies, antibody purification and protein interaction studies. G.M.H. performed MALDI–TOF studies and D.J.H. assisted with mouse studies. J.L. performed cloning, sequencing and mutagenesis of the etpA locus. M.M.O. assisted with cloning, protein purification and protein interaction studies. J.M.F. assisted with study design, protein purification, linker mutagenesis, sample preparation, scanning electron microscopy, measurement of flagella and manuscript preparation.

Author information


  1. Department of Medicine,

    • Koushik Roy
    • , Jiwen Luo
    •  & James M. Fleckenstein
  2. Department of Molecular Sciences,

    • George M. Hilliard
    •  & James M. Fleckenstein
  3. Department of Comparative Medicine, University of Tennessee Health Science Center, 956 Court Avenue, Memphis, Tennessee 38163, Tennessee, USA

    • David J. Hamilton
  4. Research Service,

    • Marguerite M. Ostmann
  5. Medicine Service, Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, Tennessee 38104, USA

    • James M. Fleckenstein


  1. Search for Koushik Roy in:

  2. Search for George M. Hilliard in:

  3. Search for David J. Hamilton in:

  4. Search for Jiwen Luo in:

  5. Search for Marguerite M. Ostmann in:

  6. Search for James M. Fleckenstein in:

Competing interests

University of Tennessee Research Foundation has applied for a patent related to studies included here.

Corresponding author

Correspondence to James M. Fleckenstein.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figures 1-6 with Legends, Supplementary Tables 1-2, Supplementary Methods and Supplementary References.

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.