Pseudomonas aeruginosa defends against phages through type IV pilus glycosylation


Since phages present a major challenge to survival in most environments, bacteria express a battery of anti-phage defences including CRISPR–Cas, restriction-modification and abortive infection systems1,2,3,4. Such strategies are effective, but the phage genome—which encodes potentially inhibitory gene products—is still allowed to enter the cell. The safest way to preclude phage infection is to block initial phage adsorption to the cell. Here, we describe a cell-surface modification that blocks infection by certain phages. Strains of the opportunistic pathogen Pseudomonas aeruginosa express one of five different type IV pilins (T4P)5, two of which are glycosylated with O-antigen units6 or polymers of d-arabinofuranose7,8,9. We propose that predation by bacteriophages that use T4P as receptors selects for strains that mask potential phage binding sites using glycosylation. Here, we show that both modifications protect P. aeruginosa from certain pilus-specific phages. Alterations to pilin sequence can also block phage infection, but glycosylation is considered less likely to create disadvantageous phenotypes. Through construction of chimeric phages, we show that specific phage tail proteins allow for infection of strains with glycosylated pili. These studies provide insight into first-line bacterial defences against predation and ways in which phages circumvent them, and provide a rationale for the prevalence of pilus glycosylation in nature.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Infection of a P. aeruginosa PAO1 pilA mutant expressing non-glycosylated versus glycosylated pilins.
Fig. 2: A chimeric phage expressing DMS3 genes in the JBD26 background gains the ability to infect a strain with glycosylated pilins.
Fig. 3: Pilin D-region sequence affects phage susceptibility.
Fig. 4: Pilin d-arabinofuranosylation blocks phage infection.


  1. 1.

    Weinbauer, M. G. Ecology of prokaryotic viruses. FEMS Microbiol. Rev. 28, 127–181 (2004).

  2. 2.

    Sampson, T. R. & Weiss, D. S. Alternative roles for CRISPR/Cas systems in bacterial pathogenesis. PLoS Pathog. 9, e1003621 (2013).

  3. 3.

    Sorek, R., Lawrence, C. M. & Wiedenheft, B. CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu. Rev. Biochem. 82, 237–266 (2013).

  4. 4.

    Labrie, S. J., Samson, J. E. & Moineau, S. Bacteriophage resistance mechanisms. Nat. Rev. Microbiol. 8, 317–327 (2010).

  5. 5.

    Kus, J. V., Tullis, E., Cvitkovitch, D. G. & Burrows, L. L. Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients. Microbiology 150, 1315–1326 (2004).

  6. 6.

    DiGiandomenico, A. et al. Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity. Mol. Microbiol. 46, 519–530 (2002).

  7. 7.

    Kus, J. V. et al. Modification of Pseudomonas aeruginosa Pa5196 type IV pilins at multiple sites with d-Araf by a novel GT-C family Arabinosyltransferase, TfpW. J. Bacteriol. 190, 7464–7478 (2008).

  8. 8.

    Harvey, H., Kus, J. V., Tessier, L., Kelly, J. & Burrows, L. L. Pseudomonas aeruginosa d-arabinofuranose biosynthetic pathway and its role in type IV pilus assembly. J. Biol. Chem. 286, 28128–28137 (2011).

  9. 9.

    Voisin, S. et al. Glycosylation of Pseudomonas aeruginosa strain Pa5196 type IV pilins with mycobacterium-like α-1,5-linked d-Araf oligosaccharides. J. Bacteriol. 189, 151–159 (2007).

  10. 10.

    Lam, J. S., Taylor, V. L., Islam, S. T., Hao, Y. & Kocincova, D. Genetic and functional diversity of Pseudomonas aeruginosa lipopolysaccharide. Front. Microbiol. 2, 118 (2011).

  11. 11.

    Bradley, D. E. & Pitt, T. L. Pilus-dependence of four Pseudomonas aeruginosa bacteriophages with non-contractile tails. J. Gen. Virol. 24, 1–15 (1974).

  12. 12.

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

  13. 13.

    Craig, L. & Li, J. Type IV pili: paradoxes in form and function. Curr. Opin. Struct. Biol. 18, 267–277 (2008).

  14. 14.

    Heiniger, R. W., Winther-Larsen, H. C., Pickles, R. J., Koomey, M. & Wolfgang, M. C. Infection of human mucosal tissue by Pseudomonas aeruginosa requires sequential and mutually dependent virulence factors and a novel pilus-associated adhesin. Cell. Microbiol. 12, 1158–1173 (2010).

  15. 15.

    Ohama, M. et al. Intratracheal immunization with pili protein protects against mortality associated with Pseudomonas aeruginosa pneumonia in mice. FEMS Immunol. Med. Microbiol. 47, 107–115 (2006).

  16. 16.

    Hahn, H. P. The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa – a review. Gene 192, 99–108 (1997).

  17. 17.

    Bucior, I., Pielage, J. F. & Engel, J. N. Pseudomonas aeruginosa pili and flagella mediate distinct binding and signaling events at the apical and basolateral surface of airway epithelium. PLoS Pathog. 8, e1002616 (2012).

  18. 18.

    James, C. E. et al. Differential infection properties of three inducible prophages from an epidemic strain of Pseudomonas aeruginosa. BMC Microbiol. 12, 216 (2012).

  19. 19.

    Bondy-Denomy, J. et al. Prophages mediate defense against phage infection through diverse mechanisms. ISME J. 10, 2854–2866 (2016).

  20. 20.

    Nguyen, Y. et al. Pseudomonas aeruginosa minor pilins prime type IVa pilus assembly and promote surface display of the PilY1 adhesin. J. Biol. Chem. 290, 601–611 (2015).

  21. 21.

    Winther-Larsen, H. C. et al. Pseudomonas aeruginosa type IV pilus expression in Neisseria gonorrhoeae: effects of pilin subunit composition on function and organelle dynamics. J. Bacteriol. 189, 6676–6685 (2007).

  22. 22.

    Giltner, C. L., Habash, M. & Burrows, L. L. Pseudomonas aeruginosa minor pilins are incorporated into type IV pili. J. Mol. Biol. 398, 444–461 (2010).

  23. 23.

    Comer, J. E., Marshall, M. A., Blanch, V. J., Deal, C. D. & Castric, P. Identification of the Pseudomonas aeruginosa 1244 pilin glycosylation site. Infect. Immun. 70, 2837–2845 (2002).

  24. 24.

    Smedley, J. G. III et al. Influence of pilin glycosylation on Pseudomonas aeruginosa 1244 pilus function. Infect. Immun. 73, 7922–7931 (2005).

  25. 25.

    Asikyan, M. L., Kus, J. V. & Burrows, L. L. Novel proteins that modulate type IV pilus retraction dynamics in Pseudomonas aeruginosa. J. Bacteriol. 190, 7022–7034 (2008).

  26. 26.

    Allison, T. M., Conrad, S. & Castric, P. The group I pilin glycan affects type IVa pilus hydrophobicity and twitching motility in Pseudomonas aeruginosa 1244. Microbiology 161, 1780–1789 (2015).

  27. 27.

    Gault, J. et al. Neisseria meningitidis type IV pili composed of sequence invariable pilins are masked by multisite glycosylation. PLoS Pathog. 11, e1005162 (2015).

  28. 28.

    Piepenbrink, K. H. et al. Structural diversity in the type IV pili of multidrug-resistant Acinetobacter. J. Biol. Chem. 291, 22924–22935 (2016).

  29. 29.

    Harvey, H., Habash, M., Aidoo, F. & Burrows, L. L. Single-residue changes in the C-terminal disulfide-bonded loop of the Pseudomonas aeruginosa type IV pilin influence pilus assembly and twitching motility. J. Bacteriol. 191, 6513–6524 (2009).

  30. 30.

    Heo, Y. J., Chung, I. Y., Choi, K. B., Lau, G. W. & Cho, Y. H. Genome sequence comparison and superinfection between two related Pseudomonas aeruginosa phages, D3112 and MP22. Microbiology 153, 2885–2895 (2007).

  31. 31.

    Castric, P., Cassels, F. J. & Carlson, R. W. Structural characterization of the Pseudomonas aeruginosa 1244 pilin glycan. J. Biol. Chem. 276, 26479–26485 (2001).

  32. 32.

    Tan, R. M. et al. Type IV pilus glycosylation mediates resistance of Pseudomonas aeruginosa to opsonic activities of the pulmonary surfactant protein A. Infect. Immun. 83, 1339–1346 (2015).

  33. 33.

    Nguyen, Y. et al. Structural and functional studies of the Pseudomonas aeruginosa minor pilin, PilE. J. Biol. Chem. 290, 26856–26865 (2015).

  34. 34.

    Craig, L. et al. 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, 1139–1150 (2003).

  35. 35.

    Kolappan, S. et al. Structure of the Neisseria meningitidis type IV pilus. Nat. Commun. 7, 13015 (2016).

  36. 36.

    Davidson, A. R., Cardarelli, L., Pell, L. G., Radford, D. R. & Maxwell, K. L. Long noncontractile tail machines of bacteriophages. Adv. Exp. Med. Biol. 726, 115–142 (2012).

  37. 37.

    Le, S. et al. Mapping the tail fiber as the receptor binding protein responsible for differential host specificity of Pseudomonas aeruginosa bacteriophages PaP1 and JG004. PLoS ONE 8, e68562 (2013).

  38. 38.

    Bondy-Denomy, J., Pawluk, A., Maxwell, K. L. & Davidson, A. R. Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429–432 (2013).

  39. 39.

    Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

  40. 40.

    Hoang, T. T., Karkhoff-Schweizer, R. R., Kutchma, A. J. & Schweizer, H. P. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212, 77–86 (1998).

  41. 41.

    Lavigne, R., Ceyssens, P. J. & Robben, J. Phage proteomics: applications of mass spectrometry. Methods Mol. Biol. 502, 239–251 (2009).

Download references


We thank K. Maxwell for helpful comments on the manuscript. This work was supported by Canadian Institutes of Health Research Open Operating Grants to L.L.B. (MOP 86639) and to A.R.D. (XNE-86943 and MOP-115039).

Author information

H.H., A.R.D. and L.L.B. designed the study; H.H., J.B.-D., H.M. and K.M.S. performed experiments; H.H., A.R.D. and L.L.B. analysed the data; A.R.D. and L.L.B. wrote the manuscript with input from H.H., J.B.-D. and H.M. All authors approved the final version.

Correspondence to Alan R. Davidson or Lori L. Burrows.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplementary Information

Supplementary Figures 1–3, Supplementary Figure References, Supplementary Tables 1–5, Supplementary Table 5 References.

Life Sciences Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Harvey, H., Bondy-Denomy, J., Marquis, H. et al. Pseudomonas aeruginosa defends against phages through type IV pilus glycosylation. Nat Microbiol 3, 47–52 (2018).

Download citation

Further reading