Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Effective assembly of fimbriae in Escherichia coli depends on the translocation assembly module nanomachine

Abstract

Outer membrane proteins are essential for Gram-negative bacteria to rapidly adapt to changes in their environment. Intricate remodelling of the outer membrane proteome is critical for bacterial pathogens to survive environmental changes, such as entry into host tissues13. Fimbriae (also known as pili) are appendages that extend up to 2 μm beyond the cell surface to function in adhesion for bacterial pathogens, and are critical for virulence. The best-studied examples of fimbriae are the type 1 and P fimbriae of uropathogenic Escherichia coli, the major causative agent of urinary tract infections in humans. Fimbriae share a common mode of biogenesis, orchestrated by a molecular assembly platform called ‘the usher’ located in the outer membrane. Although the mechanism of pilus biogenesis is well characterized, how the usher itself is assembled at the outer membrane is unclear. Here, we report that a rapid response in usher assembly is crucially dependent on the translocation assembly module. We assayed the assembly reaction for a range of ushers and provide mechanistic insight into the β-barrel assembly pathway that enables the rapid deployment of bacterial fimbriae.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Assembly of the usher FimD.
Figure 2: E. coli ΔtamA or ΔtamB mutants have defects in the assembly of FimD.
Figure 3: The TAM is required for the rapid deployment of fimbriae.
Figure 4: The TAM is required for the assembly of multiple different usher proteins.

References

  1. Vogel, J. & Papenfort, K. Small non-coding RNAs and the bacterial outer membrane. Curr. Opin. Microbiol. 9, 605–611 (2006).

    Article  CAS  PubMed  Google Scholar 

  2. Clegg, S., Wilson, J. & Johnson, J. More than one way to control hair growth: regulatory mechanisms in enterobacteria that affect fimbriae assembled by the chaperone/usher pathway. J. Bacteriol. 193, 2081–2088 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Dalebroux, Z. D. et al. Delivery of cardiolipins to the Salmonella outer membrane is necessary for survival within host tissues and virulence. Cell Host Microbe 17, 441–451 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. McMorran, L. M., Brockwell, D. J. & Radford, S. E. Mechanistic studies of the biogenesis and folding of outer membrane proteins in vitro and in vivo: what have we learned to date? Arch. Biochem. Biophys. 564, 265–280 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Selkrig, J., Leyton, D. L., Webb, C. T. & Lithgow, T. Assembly of β-barrel proteins into bacterial outer membranes. Biochim. Biophys. Acta 1843, 1542–1550 (2014).

    Article  CAS  PubMed  Google Scholar 

  6. Fleming, K. G. A combined kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria. Phil. Trans. R. Soc. Lond. B 370, 20150026 (2015).

    Article  Google Scholar 

  7. O'Neil, P. K., Rollauer, S. E., Noinaj, N. & Buchanan, S. K. Fitting the pieces of the β-barrel assembly machinery complex. Biochemistry 54, 6303–6311 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Heinz, E., Selkrig, J., Belousoff, M. J. & Lithgow, T. Evolution of the translocation and assembly module (TAM). Genome Biol. Evol. 7, 1628–1643 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Noinaj, N. et al. Structural insight into the biogenesis of β-barrel membrane proteins. Nature 501, 385–390 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Noinaj, N., Kuszak, A. J., Balusek, C., Gumbart, J. C. & Buchanan, S. K. Lateral opening and exit pore formation are required for BamA function. Structure 22, 1055–1062 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Selkrig, J. et al. Discovery of an archetypal protein transport system in bacterial outer membranes. Nature Struct. Mol. Biol. 19, 506–510 (2012).

    Article  CAS  Google Scholar 

  12. Gruss, F. et al. The structural basis of autotransporter translocation by TamA. Nature Struct. Mol. Biol. 20, 1318–1320 (2013).

    Article  CAS  Google Scholar 

  13. Shen, H. H. et al. Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes. Nature Commun. 5, 5078 (2014).

    Article  CAS  Google Scholar 

  14. Selkrig, J. et al. Conserved features in TamA enable interaction with TamB to drive the activity of the translocation and assembly module. Sci. Rep. 5, 12905 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Phan, G. et al. Crystal structure of the FimD usher bound to its cognate FimC–FimH substrate. Nature 474, 49–53 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Geibel, S., Procko, E., Hultgren, S. J., Baker, D. & Waksman, G. Structural and energetic basis of folded-protein transport by the FimD usher. Nature 496, 243–246 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Stenberg, F., von Heijne, G. & Daley, D. O. Assembly of the cytochrome bo 3 complex. J. Mol. Biol. 371, 765–773 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Noinaj, N., Rollauer, S. E. & Buchanan, S. K. The β-barrel membrane protein insertase machinery from Gram-negative bacteria. Curr. Opin. Struct. Biol. 31, 35–42 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. De Cock, H., van Blokland, S. & Tommassen, J. In vitro insertion and assembly of outer membrane protein PhoE of Escherichia coli K-12 into the outer membrane. Role of Triton X-100. J. Biol. Chem. 271, 12885–12890 (1996).

    Article  CAS  PubMed  Google Scholar 

  20. Werner, J. & Misra, R. YaeT (Omp85) affects the assembly of lipid-dependent and lipid-independent outer membrane proteins of Escherichia coli. Mol. Microbiol. 57, 1450–1459 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Korea, C. G., Ghigo, J. M. & Beloin, C. The sweet connection: Solving the riddle of multiple sugar-binding fimbrial adhesins in Escherichia coli: multiple E. coli fimbriae form a versatile arsenal of sugar-binding lectins potentially involved in surface-colonisation and tissue tropism. BioEssays: News Rev. Mol. Cell. Dev. Biol. 33, 300–311 (2011).

    Article  CAS  Google Scholar 

  22. Busch, A. & Waksman, G. Chaperone-usher pathways: diversity and pilus assembly mechanism. Phil. Trans. R. Soc. Lond. B 367, 1112–1122 (2012).

    Article  CAS  Google Scholar 

  23. Wu, X. R., Sun, T. T. & Medina, J. J. In vitro binding of type 1-fimbriated Escherichia coli to uroplakins Ia and Ib: relation to urinary tract infections. Proc. Natl Acad. Sci. USA 93, 9630–9635 (1996).

    Article  CAS  PubMed  Google Scholar 

  24. Schembri, M. A., Sokurenko, E. V. & Klemm, P. Functional flexibility of the FimH adhesin: insights from a random mutant library. Infect. Immun. 68, 2638–2646 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nilsson, L. M., Thomas, W. E., Trintchina, E., Vogel, V. & Sokurenko, E. V. Catch bond-mediated adhesion without a shear threshold: trimannose versus monomannose interactions with the FimH adhesin of Escherichia coli. J. Biol. Chem. 281, 16656–16663 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Nuccio, S. P. & Baumler, A. J. Evolution of the chaperone/usher assembly pathway: fimbrial classification goes Greek. Microbiol. Mol. Biol. Rev. 71, 551–575 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kuehn, M. J., Heuser, J., Normark, S. & Hultgren, S. J. P pili in uropathogenic E. coli are composite fibres with distinct fibrillar adhesive tips. Nature 356, 252–255 (1992).

    Article  CAS  PubMed  Google Scholar 

  28. Iguchi, A. et al. Complete genome sequence and comparative genome analysis of enteropathogenic Escherichia coli O127:H6 strain E2348/69. J. Bacteriol. 191, 347–354 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Korea, C. G., Badouraly, R., Prevost, M. C., Ghigo, J. M. & Beloin, C. Escherichia coli K-12 possesses multiple cryptic but functional chaperone-usher fimbriae with distinct surface specificities. Environ. Microbiol. 12, 1957–1977 (2010).

    Article  CAS  PubMed  Google Scholar 

  30. Wurpel, D. J., Beatson, S. A., Totsika, M., Petty, N. K. & Schembri, M. A. Chaperone-usher fimbriae of Escherichia coli. PLoS ONE 8, e52835 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Remaut, H. et al. Fiber formation across the bacterial outer membrane by the chaperone/usher pathway. Cell 133, 640–652 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Stærk, K., Khandige, S., Kolmos, H. J., Møller-Jensen, J. & Andersen, T. E. Uropathogenic Escherichia coli express type 1 fimbriae only in surface adherent populations under physiological growth conditions. J. Infect. Dis. 213, 386–394 (2016).

    Article  PubMed  Google Scholar 

  33. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  Google Scholar 

  34. Sambrook, J. & Russell, D. Molecular Cloning: A Laboratory Manual 3rd edn, Vol. 3, A2.2 (Cold Spring Harbor Laboratory Press, 2001).

    Google Scholar 

  35. Phu, L. et al. Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals. Mol. Cell. Proteom. 10, M110.003756 (2011).

    Article  Google Scholar 

  36. Wenger, C. D. & Coon, J. J. A proteomics search algorithm specifically designed for high-resolution tandem mass spectra. J. Proteome Res. 12, 1377–1386 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Schilling, B. et al. Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation. Mol. Cell Proteom. 11, 202–214 (2012).

    Article  CAS  Google Scholar 

  38. Wright, K. J., Seed, P. C. & Hultgren, S. J. Development of intracellular bacterial communities of uropathogenic Escherichia coli depends on type 1 pili. Cell Microbiol. 9, 2230–2241 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank R. Goode and O. Kleinfeld for proteomic analysis and R. Bamert, R. Dunstan, R. Grinter and E. Heinz for comments on the manuscript. The authors also thank S. Hultgren for providing strain UTI89-Ptet-fim. This work was supported by an NHMRC Program Grant (606788, to T.L. and R.A.S.) and an NHMRC Project grant (APP1042651, to M.A.S.). T.L. is an ARC Australian Laureate Fellow, MA.S. is an NHMRC Senior Research Fellow, H.H.S. is an ARC Super Science Fellow, M.J.B. is an NHMRC Biomedical Fellow, and I.D.H. is an ARC Laureate Postdoctoral Fellow.

Author information

Authors and Affiliations

Authors

Contributions

C.S., M.J.B., I.D.H., K.M.P., M.-D.P. and A.W.L. designed and carried out the analyses. C.S., M.J.B., I.D.H., J.L., M.A.S., H.H.S. and K.L.T. provided expertise for the analyses. M.A.S., M.J.B., I.D.H., J.L., R.A.S., G.W. and T.L. supervised experimental work and evaluated data. M.A.S., R.A.S., G.W. and T.L. evaluated the results and wrote the manuscript.

Corresponding author

Correspondence to Trevor Lithgow.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary Figures 1–9 (PDF 1667 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Stubenrauch, C., Belousoff, M., Hay, I. et al. Effective assembly of fimbriae in Escherichia coli depends on the translocation assembly module nanomachine. Nat Microbiol 1, 16064 (2016). https://doi.org/10.1038/nmicrobiol.2016.64

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nmicrobiol.2016.64

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing