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Architecture of the Vibrio cholerae toxin-coregulated pilus machine revealed by electron cryotomography

Abstract

Type IV pili (T4P) are filamentous appendages found on many Bacteria and Archaea. They are helical fibres of pilin proteins assembled by a multi-component macromolecular machine we call the basal body. Based on pilin features, T4P are classified into type IVa pili (T4aP) and type IVb pili (T4bP)1,2. T4aP are more widespread and are involved in cell motility3, DNA transfer4, host predation5 and electron transfer6. T4bP are less prevalent and are mainly found in enteropathogenic bacteria, where they play key roles in host colonization7. Following similar work on T4aP machines8,9, here we use electron cryotomography10 to reveal the three-dimensional in situ structure of a T4bP machine in its piliated and non-piliated states. The specific machine we analyse is the Vibrio cholerae toxin-coregulated pilus machine (TCPM). Although only about half of the components of the TCPM show sequence homology to components of the previously analysed Myxococcus xanthus T4aP machine (T4aPM), we find that their structures are nevertheless remarkably similar. Based on homologies with components of the M. xanthus T4aPM and additional reconstructions of TCPM mutants in which the non-homologous proteins are individually deleted, we propose locations for all eight TCPM components within the complex. Non-homologous proteins in the T4aPM and TCPM are found to form similar structures, suggesting new hypotheses for their functions and evolutionary histories.

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Figure 1: Visualizing the TCPM in intact V. cholerae cells.
Figure 2: Comparison between V. cholerae TCPM and M. xanthus T4aPM structures and the inferred TCPM component locations based on the T4aPM component map.
Figure 3: Structures of TCPM in ΔtcpS, ΔtcpB, ΔtcpD and ΔtcpR cells.
Figure 4: Locations of TCPM components and comparison with M. xanthus T4aPM.

References

  1. 1

    Strom, M. S. & Lory, S. Structure–function and biogenesis of the type IV pili. Annu. Rev. Microbiol. 47, 565–596 (1993).

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

    Mattick, J. S. Type IV pili and twitching motility. Annu. Rev. Microbiol. 56, 289–314 (2002).

    CAS  Article  Google Scholar 

  4. 4

    Chen, I. & Dubnau, D. DNA uptake during bacterial transformation. Nat. Rev. Microbiol. 2, 241–249 (2004).

    CAS  Article  Google Scholar 

  5. 5

    Evans, K. J., Lambert, C. & Sockett, R. E. Predation by Bdellovibrio bacteriovorus HD100 requires type IV pili. J. Bacteriol. 189, 4850–4859 (2007).

    CAS  Article  Google Scholar 

  6. 6

    Reguera, G. et al. Extracellular electron transfer via microbial nanowires. Nature 435, 1098–1101 (2005).

    CAS  Article  Google Scholar 

  7. 7

    Roux, N., Spagnolo, J. & de Bentzmann, S. Neglected but amazingly diverse type IVb pili. Res. Microbiol. 163, 659–673 (2012).

    CAS  Article  Google Scholar 

  8. 8

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

    Article  Google Scholar 

  9. 9

    Gold, V. A., Salzer, R., Averhoff, B. & Kuhlbrandt, W. Structure of a type IV pilus machinery in the open and closed state. eLife 4, e07380 (2015).

    Article  Google Scholar 

  10. 10

    Oikonomou, C. M. & Jensen, G. J. A new view into prokaryotic cell biology from electron cryotomography. Nat. Rev. Microbiol. 14, 205–220 (2016).

    CAS  Article  Google Scholar 

  11. 11

    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).

    CAS  Article  Google Scholar 

  12. 12

    Tammam, S. et al. Characterization of the PilN, PilO and PilP type IVa pilus subcomplex. Mol. Microbiol. 82, 1496–1514 (2011).

    CAS  Article  Google Scholar 

  13. 13

    Sampaleanu, L. M. et al. Periplasmic domains of pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex. J. Mol. Biol. 394, 143–159 (2009).

    CAS  Article  Google Scholar 

  14. 14

    Karuppiah, V. & Derrick, J. P. Structure of the PilM-PilN inner membrane type IV pilus biogenesis complex from Thermus thermophilus. J. Biol. Chem. 286, 24434–24442 (2011).

    CAS  Article  Google Scholar 

  15. 15

    Korotkov, K. V. et al. Structural and functional studies on the interaction of GspC and GspD in the type II secretion system. PLoS Pathogens 7, e1002228 (2011).

    CAS  Article  Google Scholar 

  16. 16

    Karuppiah, V., Collins, R. F., Thistlethwaite, A., Gao, Y. & Derrick, J. P. Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis. Proc. Natl Acad. Sci. USA 110, E4638–E4647 (2013).

    CAS  Article  Google Scholar 

  17. 17

    Karuppiah, V., Hassan, D., Saleem, M. & Derrick, J. P. Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus. Proteins 78, 2049–2057 (2010).

    CAS  PubMed  Google Scholar 

  18. 18

    Abendroth, J. et al. The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae. J. Struct. Biol. 166, 303–315 (2009).

    CAS  Article  Google Scholar 

  19. 19

    Abendroth, J., Murphy, P., Sandkvist, M., Bagdasarian, M. & Hol, W. G. The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae. J. Mol. Biol. 348, 845–855 (2005).

    CAS  Article  Google Scholar 

  20. 20

    Yamagata, A. & Tainer, J. A. Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. EMBO J. 26, 878–890 (2007).

    CAS  Article  Google Scholar 

  21. 21

    Misic, A. M., Satyshur, K. A. & Forest, K. T. P. Aeruginosa PilT structures with and without nucleotide reveal a dynamic type IV pilus retraction motor. J. Mol. Biol. 400, 1011–1021 (2010).

    CAS  Article  Google Scholar 

  22. 22

    Satyshur, K. A. et al. Crystal structures of the pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility. Structure 15, 363–376 (2007).

    CAS  Article  Google Scholar 

  23. 23

    Lu, C. et al. Hexamers of the type II secretion ATPase GspE from Vibrio cholerae with increased ATPase activity. Structure 21, 1707–1717 (2013).

    CAS  Article  Google Scholar 

  24. 24

    Kolappan, S. & Craig, L. Structure of the cytoplasmic domain of TcpE, the inner membrane core protein required for assembly of the Vibrio cholerae toxin-coregulated pilus. Acta. Crystallogr. D 69, 513–519 (2013).

    CAS  Article  Google Scholar 

  25. 25

    Krebs, S. J. & Taylor, R. K. Protection and attachment of Vibrio cholerae mediated by the toxin-coregulated pilus in the infant mouse model. J. Bacteriol. 193, 5260–5270 (2011).

    CAS  Article  Google Scholar 

  26. 26

    Taylor, R. K., Miller, V. L., Furlong, D. B. & Mekalanos, J. J. Identification of a pilus colonization factor that is coordinately regulated with cholera toxin. Ann. Sclavo. Collana Monogr. 3, 51–61 (1986).

    CAS  PubMed  Google Scholar 

  27. 27

    Manning, P. A. The tcp gene cluster of Vibrio cholerae. Gene 192, 63–70 (1997).

    CAS  Article  Google Scholar 

  28. 28

    Li, J., Egelman, E. H. & Craig, L. Structure of the Vibrio cholerae type IVb pilus and stability comparison with the Neisseria gonorrhoeae type IVa pilus. J. Mol. Biol. 418, 47–64 (2012).

    CAS  Article  Google Scholar 

  29. 29

    Taylor, R. K., Miller, V. L., Furlong, D. B. & Mekalanos, J. J. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc. Natl Acad. Sci. USA 84, 2833–2837 (1987).

    CAS  Article  Google Scholar 

  30. 30

    Gao, Y., Hauke, C. A., Marles, J. M. & Taylor, R. K. Effects of tcpB mutations on biogenesis and function of the toxin-coregulated pilus, the type IVb pilus of Vibrio cholerae. J. Bacteriol. 198, 2818–2828 (2016).

    CAS  Article  Google Scholar 

  31. 31

    Bose, N. & Taylor, R. K. Identification of a TcpC–TcpQ outer membrane complex involved in the biogenesis of the toxin-coregulated pilus of Vibrio cholerae. J. Bacteriol. 187, 2225–2232 (2005).

    CAS  Article  Google Scholar 

  32. 32

    Tripathi, S. A. & Taylor, R. K. Membrane association and multimerization of TcpT, the cognate ATPase ortholog of the Vibrio cholerae toxin-coregulated-pilus biogenesis apparatus. J. Bacteriol. 189, 4401–4409 (2007).

    CAS  Article  Google Scholar 

  33. 33

    Strom, M. S., Nunn, D. N. & Lory, S. A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Proc. Natl Acad. Sci. USA 90, 2404–2408 (1993).

    CAS  Article  Google Scholar 

  34. 34

    Iredell, J. R. & Manning, P. A. Translocation failure in a type-4 pilin operon: rfb and tcpT mutants in Vibrio cholerae. Gene 192, 71–77 (1997).

    CAS  Article  Google Scholar 

  35. 35

    Megli, C. J. & Taylor, R. K. Secretion of TcpF by the Vibrio cholerae toxin-coregulated pilus biogenesis apparatus requires an N-terminal determinant. J. Bacteriol. 195, 2718–2727 (2013).

    CAS  Article  Google Scholar 

  36. 36

    Kirn, T. J., Bose, N. & Taylor, R. K. Secretion of a soluble colonization factor by the TCP type 4 pilus biogenesis pathway in Vibrio cholerae. Mol. Microbiol. 49, 81–92 (2003).

    CAS  Article  Google Scholar 

  37. 37

    Hall, R. H., Vial, P. A., Kaper, J. B., Mekalanos, J. J. & Levine, M. M. Morphological studies on fimbriae expressed by Vibrio cholerae 01. Microb. Pathog. 4, 257–265 (1988).

    Article  Google Scholar 

  38. 38

    Kolappan, S., Ng, D., Yang, G., Harn, T. & Craig, L. Crystal structure of the minor pilin CofB, the initiator of CFA/III pilus assembly in enterotoxigenic Escherichia coli. J. Biol. Chem. 290, 25805–25818 (2015).

    CAS  Article  Google Scholar 

  39. 39

    Taniguchi, T. et al. Gene cluster for assembly of pilus colonization factor antigen III of enterotoxigenic Escherichia coli. Infect. Immun. 69, 5864–5873 (2001).

    CAS  Article  Google Scholar 

  40. 40

    Souza, D. P. et al. A component of the Xanthomonadaceae type IV secretion system combines a VirB7 motif with a N0 domain found in outer membrane transport proteins. PLoS Pathogens 7, e1002031 (2011).

    CAS  Article  Google Scholar 

  41. 41

    Berry, J. L. et al. Structure and assembly of a trans-periplasmic channel for type IV pili in Neisseria meningitidis. PLoS Pathogens 8, e1002923 (2012).

    CAS  Article  Google Scholar 

  42. 42

    Reichow, S. L., Korotkov, K. V., Hol, W. G. & Gonen, T. Structure of the cholera toxin secretion channel in its closed state. Nat. Struct. Mol. Biol. 17, 1226–1232 (2010).

    CAS  Article  Google Scholar 

  43. 43

    Tosi, T. et al. Structural similarity of secretins from type II and type III secretion systems. Structure 22, 1348–1355 (2014).

    CAS  Article  Google Scholar 

  44. 44

    Lieberman, J. A. et al. Outer membrane targeting, ultrastructure, and single molecule localization of the enteropathogenic Escherichia coli type IV pilus secretin BfpB. J. Bacteriol. 194, 1646–1658 (2012).

    CAS  Article  Google Scholar 

  45. 45

    Daniel, A. et al. Interaction and localization studies of enteropathogenic Escherichia coli type IV bundle-forming pilus outer membrane components. Microbiology 152, 2405–2420 (2006).

    CAS  Article  Google Scholar 

  46. 46

    Gomez-Duarte, O. G. et al. Genetic diversity of the gene cluster encoding longus, a type IV pilus of enterotoxigenic Escherichia coli. J. Bacteriol. 189, 9145–9149 (2007).

    CAS  Article  Google Scholar 

  47. 47

    McCallum, M. et al. PilN binding modulates the structure and binding partners of the Pseudomonas aeruginosa type IVa pilus protein PilM. J. Biol. Chem. 291, 11003-15 (2016).

    Article  Google Scholar 

  48. 48

    Bischof, L. F., Friedrich, C., Harms, A., Sogaard-Andersen, L. & van der Does, C. The type IV pilus assembly ATPase PilB of Myxococcus xanthus interacts with the inner membrane platform protein PilC and the nucleotide-binding protein PilM. J. Biol. Chem. 291, 6946–6957 (2016).

    CAS  Article  Google Scholar 

  49. 49

    Friedrich, C., Bulyha, I. & Sogaard-Andersen, L. Outside-in assembly pathway of the type IV pilus system in Myxococcus xanthus. J. Bacteriol. 196, 378–390 (2014).

    Article  Google Scholar 

  50. 50

    Tivol, W. F., Briegel, A. & Jensen, G. J. An improved cryogen for plunge freezing. Microsc. Microanal. 14, 375–379 (2008).

    CAS  Article  Google Scholar 

  51. 51

    Zheng, S. Q. et al. UCSF tomography: an integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction. J. Struct. Biol. 157, 138–147 (2007).

    CAS  Article  Google Scholar 

  52. 52

    Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    CAS  Article  Google Scholar 

  53. 53

    Agulleiro, J. I. & Fernandez, J. J. Fast tomographic reconstruction on multicore computers. Bioinformatics 27, 582–583 (2011).

    CAS  Article  Google Scholar 

  54. 54

    Nicastro, D. et al. The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313, 944–948 (2006).

    CAS  Article  Google Scholar 

  55. 55

    Ulrich, L. E. & Zhulin, I. B. The MiST2 database: a comprehensive genomics resource on microbial signal transduction. Nucleic Acids Res. 38, D401–D407 (2010).

    CAS  Article  Google Scholar 

  56. 56

    Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinformatics 10, 421 (2009).

    Article  Google Scholar 

  57. 57

    Adebali, O., Ortega, D. R. & Zhulin, I. B. CDvist: a webserver for identification and visualization of conserved domains in protein sequences. Bioinformatics 31, 1475–1477 (2015).

    CAS  Article  Google Scholar 

  58. 58

    Soding, J. Protein homology detection by HMM-HMM comparison. Bioinformatics 21, 951–960 (2005).

    Article  Google Scholar 

  59. 59

    Juncker, A. S. et al. Prediction of lipoprotein signal peptides in Gram-negative bacteria. Protein Sci. 12, 1652–1662 (2003).

    CAS  Article  Google Scholar 

  60. 60

    Kawahara, K. et al. Homo-trimeric structure of the type IVb minor pilin CofB suggests mechanism of CFA/III pilus assembly in human enterotoxigenic Escherichia coli. J. Mol. Biol. 428, 1209–1226 (2016).

    CAS  Article  Google Scholar 

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Acknowledgements

The authors thank C. Oikonomou and C. Shaffer for discussions and editorial assistance. This work was supported by NIH grant R01 AI127401 to G.J.J., the Howard Hughes Medical Institute and the John Templeton Foundation as part of the Boundaries of Life project. The opinions expressed in this publication are those of the authors and do not necessarily reflect the views of the John Templeton Foundation.

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Y.-W.C. and A.K. collected, processed and analysed the ECT data. D.R.O. performed the bioinformatics analyses. L.A.R. assisted with ECT data processing. G.K., J.A.S. and R.K.T. provided the V. cholerae strains. Y.-W.C., A.K., D.R.O. and G.J.J. wrote the paper.

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Correspondence to Grant J. Jensen.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Discussion, Supplementary Figures 1–7, Supplementary Tables 1–3, Supplementary References. (PDF 14686 kb)

Supplementary Video 1

Comparison of the TCPM conformation in piliated and non-piliated states. Conformational differences between piliated and non-piliated TCPMs are revealed by morphing from one density to another. (MOV 575 kb)

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Chang, YW., Kjær, A., Ortega, D. et al. Architecture of the Vibrio cholerae toxin-coregulated pilus machine revealed by electron cryotomography. Nat Microbiol 2, 16269 (2017). https://doi.org/10.1038/nmicrobiol.2016.269

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