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Real-time microscopy and physical perturbation of bacterial pili using maleimide-conjugated molecules

Abstract

Bacteria use surface-exposed, proteinaceous fibers called pili for diverse behaviors, including horizontal gene transfer, surface sensing, motility, and pathogenicity. Visualization of these filamentous nanomachines and their activity in live cells has proven challenging, largely due to their small size. Here, we describe a broadly applicable method for labeling and imaging pili and other surface-exposed nanomachines in live cells. This technique uses a combination of genetics and maleimide-based click chemistry in which a cysteine substitution is made in the major pilin subunit for subsequent labeling with thiol-reactive maleimide dyes. Large maleimide-conjugated molecules can also be used to physically interfere with the dynamic activity of filamentous nanomachines. We describe parameters for selecting cysteine substitution positions, optimized labeling conditions for epifluorescence imaging of pilus fibers, and methods for impeding pilus activity. After cysteine knock-in strains have been generated, this protocol can be completed within 30 min to a few hours, depending on the species and the experiment of choice. Visualization of extracellular nanomachines such as pili using this approach can provide a more comprehensive understanding of the role played by these structures in distinct bacterial behaviors.

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Fig. 1: Pili can be visualized with maleimide-reactive fluorescent dyes.
Fig. 2: Accessible residues are unique to each pilin.
Fig. 3: Cysteine substitution and fluorescent maleimide labeling allows visualization of different classes of pili.
Fig. 4: Observation of pili is affected by multiple variables.
Fig. 5: Pilus retraction can be blocked using different maleimide conjugates.

Data availability

The data that support this study are available from the corresponding author upon reasonable request.

References

  1. Green, E. R. & Mecsas, J. Bacterial secretion systems: an overview. in Virulence Mechanisms of Bacterial Pathogens 5th edn (eds Kudva, I. T. et al.) 215–239 (2016).

  2. Kearns, D. B. A field guide to bacterial swarming motility. Nat. Rev. Microbiol. 8, 634–644 (2010).

    Article  CAS  Google Scholar 

  3. Pelicic, V. et al. Type IV pili: e pluribus unum? Microbiology 68, 827–837 (2003).

    Google Scholar 

  4. Berry, J.-L. & Pelicic, V. Exceptionally widespread nanomachines composed of type IV pilins: the prokaryotic Swiss Army knives. FEMS Microbiol. Rev. 39, 134–154 (2015).

    Article  CAS  Google Scholar 

  5. Blair, K. M., Turner, L., Winkelman, J. T., Berg, H. C. & Kearns, D. B. A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science 320, 1636–1638 (2008).

    Article  CAS  Google Scholar 

  6. Ellison, C. K. et al. Obstruction of pilus retraction stimulates bacterial surface sensing. Science 358, 535–538 (2017).

    Article  CAS  Google Scholar 

  7. Paradis, G. et al. Variability in bacterial flagella re-growth patterns after breakage. Sci. Rep. 7, 1282 (2017).

    Article  Google Scholar 

  8. Ellison, C. K. et al. Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae. Nat. Microbiol. 3, 773–780 (2018).

    Article  CAS  Google Scholar 

  9. Berne, C. et al. Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA. Mol. Microbiol. 110, 219–238 (2018).

    Article  CAS  Google Scholar 

  10. Cairns, L. S. et al. FlgN is required for flagellum-based motility by Bacillus subtilis. J. Bacteriol. 196, 2216–2226 (2014).

    Article  Google Scholar 

  11. Turner, L., Stern, A. S. & Berg, H. C. Growth of flagellar filaments of Escherichia coli is independent of filament length. J. Bacteriol. 194, 2437–2442 (2012).

    Article  CAS  Google Scholar 

  12. Dietrich, M., Mollenkopf, H., So, M. & Friedrich, A. Pilin regulation in the pilT mutant of Neisseria gonorrhoeae strain MS11. FEMS Microbiol. Lett. 296, 248–256 (2009).

    Article  CAS  Google Scholar 

  13. Blocker, A., Komoriya, K. & Aizawa, S.-I. Type III secretion systems and bacterial flagella: insights into their function from structural similarities. Proc. Natl. Acad. Sci. USA 100, 3027–3030 (2003).

    Article  CAS  Google Scholar 

  14. Craig, L. et al. Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions. Mol. Cell 23, 651–662 (2006).

    Article  CAS  Google Scholar 

  15. Chang, Y.-W. et al. Architecture of the type IVa pilus machine. Science 351, aad2001 (2016).

    Article  Google Scholar 

  16. Wang, F. et al. Cryoelectron microscopy reconstructions of the Pseudomonas aeruginosa and Neisseria gonorrhoeae type IV pili at sub-nanometer resolution. Structure 25, 1423–1435.e4 (2017).

    Article  CAS  Google Scholar 

  17. Mahmoud, K. K. & Koval, S. F. Characterization of type IV pili in the life cycle of the predator bacterium Bdellovibrio. Microbiology 156, 1040–1051 (2010).

    Article  CAS  Google Scholar 

  18. Seitz, P. & Blokesch, M. DNA-uptake machinery of naturally competent Vibrio cholerae. Proc. Natl. Acad. Sci. USA 110, 17987–17992 (2013).

    Article  CAS  Google Scholar 

  19. Imhaus, A.-F. & Duménil, G. The number of Neisseria meningitidis type IV pili determines host cell interaction. EMBO J. 33, 1767–1783 (2014).

    Article  CAS  Google Scholar 

  20. Skerker, J. M. & Berg, H. C. Direct observation of extension and retraction of type IV pili. Proc. Natl. Acad. Sci. USA 98, 6901–6904 (2001).

    Article  CAS  Google Scholar 

  21. Skerker, J. M. & Shapiro, L. Identification and cell cycle control of a novel pilus system in Caulobacter crescentus. EMBO J. 19, 3223–3234 (2000).

    Article  CAS  Google Scholar 

  22. Bernard, C. S., Bordi, C., Termine, E., Filloux, A. & de Bentzmann, S. Organization and PprB-dependent control of the Pseudomonas aeruginosa tad Locus, involved in Flp pilus biology. J. Bacteriol. 191, 1961–1973 (2009).

    Article  CAS  Google Scholar 

  23. Turner, L. & Berg, H. C. Labeling bacterial flagella with fluorescent dyes. Methods Mol. Biol. 1729, 71–76 (2018).

    Article  CAS  Google Scholar 

  24. Giltner, C. L., Nguyen, Y. & Burrows, L. L. Type IV pilin proteins: versatile molecular modules. Microbiol. Mol. Biol. Rev. 76, 740–772 (2012).

    Article  CAS  Google Scholar 

  25. Craig, L., Pique, M. E. & Tainer, J. A. Type IV pilus structure and bacterial pathogenicity. Nat. Rev. Microbiol. 2, 363–378 (2004).

    Article  CAS  Google Scholar 

  26. Petersen, B. et al. A generic method for assignment of reliability scores applied to solvent accessibility predictions. BMC Struct. Biol. 9, 51 (2009).

    Article  Google Scholar 

  27. Ng, D. et al. The Vibrio cholerae minor pilin TcpB initiates assembly and retraction of the toxin-coregulated pilus. PLOS Pathog. 12, e1006109 (2016).

    Article  Google Scholar 

  28. Jones, C. J. et al. C-di-GMP regulates motile to sessile transition by modulating MshA pili biogenesis and near-surface motility behavior in Vibrio cholerae. PLoS Pathog. 11, e1005068 (2015).

    Article  Google Scholar 

  29. Burrows, L. L. Twitching motility: type IV pili in action. Annu. Rev. Microbiol. 66, 493–520 (2012).

    Article  CAS  Google Scholar 

  30. Marks, M. E. et al. The genetic basis of laboratory adaptation in Caulobacter crescentus. J. Bacteriol. 192, 3678–3688 (2010).

    Article  CAS  Google Scholar 

  31. Miller, V. L., DiRita, V. J. & Mekalanos, J. J. Identification of toxS, a regulatory gene whose product enhances toxR-mediated activation of the cholera toxin promoter. J. Bacteriol. 171, 1288–1293 (1989).

    Article  CAS  Google Scholar 

  32. Ried, J. L. & Collmer, A. An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in gram-negative bacteria by marker exchange-eviction mutagenesis. Gene 57, 239–246 (1987).

    Article  CAS  Google Scholar 

  33. Dalia, A. B., McDonough, E. & Camilli, A. Multiplex genome editing by natural transformation. Proc. Natl. Acad. Sci. USA 111, 8937–8942 (2014).

    Article  CAS  Google Scholar 

  34. Dalia, T. N. et al. Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases. Nucleic Acids Res. 45, 7527–7537 (2017).

    Article  CAS  Google Scholar 

  35. Dalia, A. B. Natural cotransformation and multiplex genome editing by natural transformation (MuGENT) of Vibrio cholerae. Methods Mol. Biol. 1839, 53–64 (2018).

    Article  Google Scholar 

  36. POINDEXTER, J. S. Biological properties and classification of the Caulobacter group. Bacteriol. Rev. 28, 231–295 (1964).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Hepp, C. & Maier, B. Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism. Proc. Natl. Acad. Sci. USA 113, 12467–12472 (2016).

    Article  CAS  Google Scholar 

  38. Pönisch, W. et al. Pili mediated intercellular forces shape heterogeneous bacterial microcolonies prior to multicellular differentiation. Sci. Rep. 8, 16567 (2018).

    Article  Google Scholar 

  39. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).

    Article  CAS  Google Scholar 

  40. Craig, L. et al. Type IV pilin structure and assembly. Mol. Cell 11, 1139–1150 (2003).

    Article  CAS  Google Scholar 

  41. Pettersen, E. F. et al. UCSF Chimera? A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. D. Koch, A. M. Randich, and M. Jacq for critical feedback on the manuscript. This work was supported by grant R35GM122556 from the National Institutes of Health and by a Canada 150 Research Chair in Bacterial Cell Biology to Y.V.B., by grants R35GM12867 and AI118863 from the National Institutes of Health to A.B.D., and by National Science Foundation Fellowship 1342962 to C.K.E.

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C.K.E. and Y.V.B conceived the study. C.K.E. and T.N.D. performed the experiments. C.K.E., A.B.D., and Y.V.B. analyzed the data. C.K.E. wrote the manuscript with help from A.B.D. and Y.V.B.

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Correspondence to Yves V. Brun.

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Ellison, C. K. et al. Science 358, 535–538 (2017): http://science.sciencemag.org/content/358/6362/535

Ellison, C. K. et al. Nat. Microbiol. 3, 773–780 (2018): https://www.nature.com/articles/s41564-018-0174-y

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Ellison, C.K., Dalia, T.N., Dalia, A.B. et al. Real-time microscopy and physical perturbation of bacterial pili using maleimide-conjugated molecules. Nat Protoc 14, 1803–1819 (2019). https://doi.org/10.1038/s41596-019-0162-6

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