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.

  • Article
  • Published:

Cyclic-di-GMP regulates lipopolysaccharide modification and contributes to Pseudomonas aeruginosa immune evasion

Nature Microbiology volume 2, Article number: 17027 (2017) Cite this article

Subjects

Abstract

Pseudomonas aeruginosa is a Gram-negative bacterial pathogen associated with acute and chronic infections. The universal cyclic-di-GMP second messenger is instrumental in the switch from a motile lifestyle to resilient biofilm as in the cystic fibrosis lung. The SadC diguanylate cyclase is associated with this patho-adaptive transition. Here, we identify an unrecognized SadC partner, WarA, which we show is a methyltransferase in complex with a putative kinase, WarB. We established that WarA binds to cyclic-di-GMP, which potentiates its methyltransferase activity. Together, WarA and WarB have structural similarities with the bifunctional Escherichia coli lipopolysaccharide (LPS) O antigen regulator WbdD. Strikingly, WarA influences P. aeruginosa O antigen modal distribution and interacts with the LPS biogenesis machinery. LPS is known to modulate the immune response in the host, and by using a zebrafish infection model, we implicate WarA in the ability of P. aeruginosa to evade detection by the host.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: WarA interacts with SadC.
Figure 2: Structural homology of WbdD and WarAB.
Figure 3: WarA impact LPS modal distribution.
Figure 4: WarA is a functional methyltransferase and binds c-di-GMP.
Figure 5: Role of SadC and WarA in infection and immune evasion.
Figure 6: Role of WarA in CPA biosynthesis.

Similar content being viewed by others

References

  1. Moscoso, J. A., Mikkelsen, H., Heeb, S., Williams, P. & Filloux, A. The Pseudomonas aeruginosa sensor RetS switches type III and type VI secretion via c-di-GMP signalling. Environ. Microbiol. 13, 3128–3138 (2011).

    Article  CAS  Google Scholar 

  2. Lam, M. Y. et al. Occurrence of a common lipopolysaccharide antigen in standard and clinical strains of Pseudomonas aeruginosa. J. Clin. Microbiol. 27, 962–967 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Islam, S. T. & Lam, J. S. Synthesis of bacterial polysaccharides via the Wzx/Wzy-dependent pathway. Can. J. Microbiol. 60, 697–716 (2014).

    Article  CAS  Google Scholar 

  4. Bellini, D. et al. Crystal structure of an HD-GYP domain cyclic-di-GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre. Mol. Microbiol. 91, 26–38 (2014).

    Article  CAS  Google Scholar 

  5. Ryan, R. P. Cyclic di-GMP signalling and the regulation of bacterial virulence. Microbiology 159(Pt 7), 1286–1297 (2013).

    Article  CAS  Google Scholar 

  6. Sundriyal, A. et al. Inherent regulation of EAL domain-catalyzed hydrolysis of second messenger cyclic di-GMP. J. Biol. Chem. 289, 6978–6990 (2014).

    Article  CAS  Google Scholar 

  7. Romling, U., Galperin, M. Y. & Gomelsky, M. Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol. Mol. Biol. Rev. 77, 1–52 (2013).

    Article  Google Scholar 

  8. Mulcahy, H. et al. Pseudomonas aeruginosa RsmA plays an important role during murine infection by influencing colonization, virulence, persistence, and pulmonary inflammation. Infect. Immun. 76, 632–638 (2008).

    Article  CAS  Google Scholar 

  9. Moscoso, J. A. et al. The diguanylate cyclase SadC is a central player in Gac/Rsm-mediated biofilm formation in Pseudomonas aeruginosa. J. Bacteriol. 196, 4081–4088 (2014).

    Article  Google Scholar 

  10. Houot, L., Fanni, A., de Bentzmann, S. & Bordi, C. A bacterial two-hybrid genome fragment library for deciphering regulatory networks of the opportunistic pathogen Pseudomonas aeruginosa. Microbiology 158(Pt_8), 1964–1971 (2012).

    Article  CAS  Google Scholar 

  11. Wu, Y., Li, Q. & Chen, X. Z. Detecting protein–protein interactions by far western blotting. Nat. Protoc. 2, 3278–3284 (2007).

    Article  CAS  Google Scholar 

  12. 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 

  13. Hagelueken, G. et al. Structure of WbdD: a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coli O9a. Mol. Microbiol. 86, 730–742 (2012).

    Article  CAS  Google Scholar 

  14. Yokota, S., Kaya, S., Kawamura, T., Araki, Y. & Ito, E. The structure of the O-specific chain of lipopolysaccharide from Pseudomonas aeruginosa IID 1008 (ATCC 27584). J. Biochem. 99, 1551–1561 (1986).

    Article  CAS  Google Scholar 

  15. Arsenault, T. L. et al. Structural studies on the polysaccharide portion of ‘A-band’ lipopolysaccharide from a mutant (AK1401) of Pseudomonas aeruginosa strain PAO1. Can. J. Chem. 69, 1273–1280 (1991).

    Article  CAS  Google Scholar 

  16. Hao, Y., King, J. D., Huszczynski, S., Kocincova, D. & Lam, J. S. Five new genes are important for common polysaccharide antigen biosynthesis in Pseudomonas aeruginosa. mBio 4, e00631-12 (2013).

    Article  Google Scholar 

  17. Clarke, B. R., Greenfield, L. K., Bouwman, C. & Whitfield, C. Coordination of polymerization, chain termination, and export in assembly of the Escherichia coli lipopolysaccharide O9a antigen in an ATP-binding cassette transporter-dependent pathway. J. Biol. Chem. 284, 30662–30672 (2009).

    Article  CAS  Google Scholar 

  18. Clarke, B. R., Cuthbertson, L. & Whitfield, C. Nonreducing terminal modifications determine the chain length of polymannose O antigens of Escherichia coli and couple chain termination to polymer export via an ATP-binding cassette transporter. J. Biol. Chem. 279, 35709–35718 (2004).

    Article  CAS  Google Scholar 

  19. Bélanger, M., Burrows, L. L. & Lam, J. S. Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide. Microbiology 145(Pt 12), 3505–3521 (1999).

    Article  Google Scholar 

  20. Rocchetta, H. L., Burrows, L. L. & Lam, J. S. Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa. Microbiol. Mol. Biol. Rev. 63, 523–553 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Homonylo, M. K., Wilmot, S. J., Lam, J. S., MacDonald, L. A. & Whitfield, C. Monoclonal antibodies against the capsular K antigen of Escherichia coli (O9:K30(A):H12): characterisation and use in analysis of K antigen organisation on the cell surface. Can. J. Microbiol. 34, 1159–1165 (1988).

    Article  CAS  Google Scholar 

  22. Hagelueken, G. et al. A coiled-coil domain acts as a molecular ruler to regulate O-antigen chain length in lipopolysaccharide. Nat. Struct. Mol. Biol. 22, 50–56 (2014).

    Article  Google Scholar 

  23. Liston, S. D. et al. Domain interactions control complex formation and polymerase specificity in the biosynthesis of the Escherichia coli O9a antigen. J. Biol. Chem. 290, 1075–1085 (2015).

    Article  CAS  Google Scholar 

  24. King, J. D., Kocincova, D., Westman, E. L. & Lam, J. S. Review: lipopolysaccharide biosynthesis in Pseudomonas aeruginosa. Innate Immun. 15, 261–312 (2009).

    Article  CAS  Google Scholar 

  25. Hao, Y., Murphy, K., Lo, R. Y., Khursigara, C. M. & Lam, J. S. Single-nucleotide polymorphisms found in the migA and wbpX glycosyltransferase genes account for the intrinsic lipopolysaccharide defects exhibited by Pseudomonas aeruginosa PA14. J. Bacteriol. 197, 2780–2791 (2015).

    Article  CAS  Google Scholar 

  26. Wang, S. et al. Biosynthesis of the common polysaccharide antigen of Pseudomonas aeruginosa PAO1: characterization and role of GDP-d-rhamnose:glcNAc/GalNAc-diphosphate-lipid α1,3-d-rhamnosyltransferase WbpZ. J. Bacteriol. 197, 2012–2019 (2015).

    Article  CAS  Google Scholar 

  27. Roelofs, K. G., Wang, J., Sintim, H. O. & Lee, V. T. Differential radial capillary action of ligand assay for high-throughput detection of protein–metabolite interactions. Proc. Natl Acad. Sci. USA 108, 15528–15533 (2011).

    Article  CAS  Google Scholar 

  28. Trampari, E. et al. Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP. J. Biol. Chem. 290, 24470–24483 (2015).

    Article  CAS  Google Scholar 

  29. Christen, B. et al. Allosteric control of cyclic di-GMP signaling. J. Biol. Chem. 281, 32015–32024 (2006).

    Article  CAS  Google Scholar 

  30. Pier, G. B. Pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity. Int. J. Med. Microbiol. 297, 277–295 (2007).

    Article  CAS  Google Scholar 

  31. Cryz, S. J. Jr, Pitt, T. L., Fürer, E. & Germanier, R. Role of lipopolysaccharide in virulence of Pseudomonas aeruginosa. Infect. Immun. 44, 508–513 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Goldberg, J. B., Coyne, M. J. Jr, Neely, A. N. & Holder, I. A. Avirulence of a Pseudomonas aeruginosa algC mutant in a burned-mouse model of infection. Infect. Immun. 63, 4166–4169 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Priebe, G. P. et al. The galU gene of Pseudomonas aeruginosa is required for corneal infection and efficient systemic spread following pneumonia but not for infection confined to the lung. Infect. Immun. 72, 4224–4232 (2004).

    Article  CAS  Google Scholar 

  34. Peterman, E. M. et al. Neutralization of mitochondrial superoxide by superoxide dismutase 2 promotes bacterial clearance and regulates phagocyte numbers in zebrafish. Infect. Immun. 83, 430–440 (2015).

    Article  CAS  Google Scholar 

  35. Joyce, S. A. & Gahan, C. G. Molecular pathogenesis of Listeria monocytogenes in the alternative model host Galleria mellonella. Microbiology 156, 3456–3468 (2010).

    Article  CAS  Google Scholar 

  36. Clatworthy, A. E. et al. Pseudomonas aeruginosa infection of zebrafish involves both host and pathogen determinants. Infect. Immun. 77, 1293–1303 (2009).

    Article  CAS  Google Scholar 

  37. Hall, C., Flores, M. V., Storm, T., Crosier, K. & Crosier, P. The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev. Biol. 7, 42 (2007).

    Article  Google Scholar 

  38. Amikam, D. & Benziman, M. Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens. J. Bacteriol. 171, 6649–6655 (1989).

    Article  CAS  Google Scholar 

  39. Mayer, R. et al. Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants. Proc. Natl Acad. Sci. USA 88, 5472–5476 (1991).

    Article  CAS  Google Scholar 

  40. Amikam, D. & Galperin, M. Y. Pilz domain is part of the bacterial c-di-GMP binding protein. Bioinformatics 22, 3–6 (2006).

    Article  CAS  Google Scholar 

  41. Roelofs, K. G. et al. Systematic identification of cyclic-di-GMP binding proteins in Vibrio cholerae reveals a novel class of cyclic-di-GMP-binding ATPases associated with type II secretion systems. PLoS Pathog. 11, e1005232 (2015).

    Article  Google Scholar 

  42. Corrigan, R. M., Abbott, J. C., Burhenne, H., Kaever, V. & Gründling, A. c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress. PLoS Pathog. 7, e1002217 (2011).

    Article  CAS  Google Scholar 

  43. Corrigan, R. M. et al. Systematic identification of conserved bacterial c-di-AMP receptor proteins. Proc. Natl Acad. Sci. USA 110, 9084–9089 (2013).

    Article  CAS  Google Scholar 

  44. Lori, C. et al. Cyclic di-GMP acts as a cell cycle oscillator to drive chromosome replication. Nature 523, 236–239 (2015).

    Article  CAS  Google Scholar 

  45. Ojeniyi, B., Lam, J. S., Høiby, N. & Rosdahl, V. T. A comparison of the efficiency in serotyping of Pseudomonas aeruginosa from cystic fibrosis patients using monoclonal and polyclonal antibodies. APMIS 97, 631–636 (1989).

    Article  CAS  Google Scholar 

  46. Sezonov, G., Joseleau-Petit, D. & D'Ari, R. Escherichia coli physiology in Luria–Bertani broth. J. Bacteriol. 189, 8746–8749 (2007).

    Article  CAS  Google Scholar 

  47. Miller, J. H. Experiments in Molecular Genetics (Cold Spring Harbor Laboratory Press, 1972).

    Google Scholar 

  48. Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd edn (Cold Spring Harbor Laboratory Press, 1989).

    Google Scholar 

  49. Pessi, G. & Haas, D. Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa. J. Bacteriol. 182, 6940–6949 (2000).

    Article  CAS  Google Scholar 

  50. Bordi, C. et al. Regulatory RNAs and the HptB/RetS signalling pathways fine-tune Pseudomonas aeruginosa pathogenesis. Mol. Microbiol. 76, 1427–1443 (2010).

    Article  CAS  Google Scholar 

  51. Karimova, G., Pidoux, J., Ullmann, A. & Ladant, D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc. Natl Acad. Sci. USA 95, 5752–5756 (1998).

    Article  CAS  Google Scholar 

  52. Hitchcock, P. J. & Brown, T. M. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154, 269–277 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Fomsgaard, A., Freudenberg, M. A. & Galanos, C. Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels. J. Clin. Microbiol. 28, 2627–2631 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. de Kievit, T. R., Dasgupta, T., Schweizer, H. & Lam, J. S. Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype O5). Mol. Microbiol. 16, 565–574 (1995).

    Article  CAS  Google Scholar 

  55. Blake, M. S., Johnston, K. H., Russell-Jones, G. J. & Gotschlich, E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on western blots. Anal. Biochem. 136, 175–179 (1984).

    Article  CAS  Google Scholar 

  56. Harding, C. R., Schroeder, G. N., Collins, J. W. & Frankel, G. Use of Galleria mellonella as a model organism to study Legionella pneumophila infection. J. Vis. Exp. 81, e50964 (2013).

    Google Scholar 

  57. Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio) (Univ. Oregon Press, 1993).

    Google Scholar 

  58. Rocker, A. J., Weiss, A. R., Lam, J. S., Van Raay, T. J. & Khursigara, C. M. Visualizing and quantifying Pseudomonas aeruginosa infection in the hindbrain ventricle of zebrafish using confocal laser scanning microscopy. J. Microbiol. Methods 117, 85–94 (2015).

    Article  Google Scholar 

  59. Mostowy, S. et al. The zebrafish as a new model for the in vivo study of Shigella flexneri interaction with phagocytes and bacterial autophagy. PLoS Pathog. 9, e1003588 (2013).

    Article  CAS  Google Scholar 

  60. Mazon Moya, M. J., Colluci-Guyon, E. & Mostowy, S. Use of Shigella flexneri to study autophagy–cytoskeleton interactions. J. Vis. Exp. 91, e51601 (2014).

    Google Scholar 

  61. Stockhammer, O. W., Zakrzewska, A., Hegedus, Z., Spaink, H. P. & Meijer, A. H. Transcriptome profiling and functional analyses of the zebrafish embryonic innate immune response to Salmonella infection. J. Immunol. 182, 5641–5653 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Work in A. Filloux's laboratory was supported by the BBSRC (grant BB/L007959/1). Work in the Mostowy laboratory is supported by a Wellcome Trust Research Career Development Fellowship (WT097411MA) and the Lister Institute of Preventive Medicine. Work in the JSL laboratory is supported by an operating grant from the Canadian Institutes of Health Research (MOP-14687). Y.H. is a recipient of a postdoctoral fellowship from Cystic Fibrosis Canada and J.S.L. holds a Canada Research Chair in Cystic Fibrosis and Microbial Glycobiology funded by the Canadian Foundation of Innovation. The authors thank M. Jun for construction of plasmids pUT18C_wbpX, pUT18C_wbpY and pUT18C_wbpZ and for testing them in the BTH system. The authors thank V. Lee for the gift of the plasmid to express and purify WspR, A. Mylona for the gift of plasmid pET-41a-3CD and A. Grundling for the provision of unlabelled nucleotides.

Author information

Author notes

  1. Joana A. Moscoso

    Present address: †Present address: Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200 Porto, Portugal; the Group of Molecular Microbiology, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal.,

Authors and Affiliations

  1. Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, SW7 2AZ, UK

    Ronan R. McCarthy, Joana A. Moscoso & Alain Filloux

  2. Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, SW7 4AZ, UK

    Maria J. Mazon-Moya & Serge Mostowy

  3. Department of Molecular and Cellular Biology, University of Guelph, Guelph, N1G 2W1, Ontario, Canada

    Youai Hao & Joseph S. Lam

  4. Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, Aix-Marseille Université, CNRS UMR7255, Marseille, France

    Christophe Bordi

Authors
  1. Ronan R. McCarthy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria J. Mazon-Moya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joana A. Moscoso
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Youai Hao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joseph S. Lam
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christophe Bordi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Serge Mostowy
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Alain Filloux
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.F. and R.M.C. conceived and designed the experiments, and wrote the paper. J.S.L. and Y.H. contributed materials, including anti-LPS monoclonal antibodies, performed LPS analysis and helped with editing the manuscript. J.A.M. and C.B. performed the BTH screen. M.J.M.-M. and S.M. contributed zebrafish experiments. R.M.C. conducted all experiments.

Corresponding author

Correspondence to Alain Filloux.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1–9 and Supplementary Tables 1 and 2. (PDF 1737 kb)

Supplementary Video 1

PAK::gfp neutrophil recruitment to site of infection. Tg(lyz:dsRed)nz50 zebrafish larvae infected in the hindbrain ventricle with 5 × 104 CFU of PAK::gfp and imaged every 10 minutes for 12 hours using fluorescent microscopy. (AVI 908 kb)

Supplementary Video 2

PAK∆sadC::gfp neutrophil recruitment to site of infection. Tg(lyz:dsRed)nz50 zebrafish larvae infected in the hindbrain ventricle with 5 × 104 CFU of PAK∆sadC::gfp and imaged every 10 minutes for 12 hours using fluorescent microscopy. (AVI 1259 kb)

Supplementary Video 3

PAK∆warA::gfp neutrophil recruitment to site of infection. Tg(lyz:dsRed)nz50 zebrafish larvae infected in the hindbrain ventricle with 5 × 104 CFU of PAK∆warA::gfp and imaged every 10 minutes for 12 hours using fluorescent microscopy. (AVI 1060 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

McCarthy, R., Mazon-Moya, M., Moscoso, J. et al. Cyclic-di-GMP regulates lipopolysaccharide modification and contributes to Pseudomonas aeruginosa immune evasion. Nat Microbiol 2, 17027 (2017). https://doi.org/10.1038/nmicrobiol.2017.27

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nmicrobiol.2017.27

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

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