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:

Respiratory protein–generated reactive oxygen species as an antimicrobial strategy

Nature Immunology volume 8, pages 1114–1122 (2007)Cite this article

Abstract

The evolution of the host-pathogen relationship comprises a series of invasive-defensive tactics elicited by both participants. The stereotype is that the antimicrobial immune response requires multistep processes. Little is known about the primordial immunosurveillance system, which probably has components that directly link sensors and effectors. Here we found that the respiratory proteins of both the horseshoe crab and human were directly activated by microbial proteases and were enhanced by pathogen-associated molecular patterns, resulting in the production of more reactive oxygen species. Hemolytic virulent pathogens, which produce proteases as invasive factors, are more susceptible to this killing mechanism. This 'shortcut' antimicrobial strategy represents a fundamental and universal mode of immunosurveillance, which has been in existence since before the split of protostomes and deuterostomes and still persists today.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Activation of HMC-PPO to PO by microbial proteases and PAMPs.
Figure 2: In vitro antimicrobial action of activated PO.
Figure 3: PO triggered by microbial protease contributes to in vivo bacterial clearance.
Figure 4: Pseudoperoxidase activity of metHb is triggered synergistically by microbial proteases and PAMPs.
Figure 5: Functional evaluation of the metHb-mediated ROS production in RBC by various bacteria.

Similar content being viewed by others

References

  1. Kuehn, M.J. & Kesty, N.C. Bacterial outer membrane vesicles and the host-pathogen interaction. Genes Dev. 19, 2645–2655 (2005).

    Article  CAS  Google Scholar 

  2. Winzer, K. & Williams, P. Quorum sensing and the regulation of virulence gene expression in pathogenic bacteria. Int. J. Med. Microbiol. 291, 131–143 (2001).

    Article  CAS  Google Scholar 

  3. Costerton, J.W., Stewart, P.S. & Greenberg, E.P. Bacterial biofilm: a common cause of persistent infections. Science 284, 1318–1322 (1999).

    Article  CAS  Google Scholar 

  4. Murphy, P.M. Viral exploitation and subversion of the immune system through chemokine mimicry. Nat. Immunol. 2, 116–122 (2001).

    Article  CAS  Google Scholar 

  5. Ganz, T. & Lehrer, R.I. Antimicrobial peptides in mammalian and insect host defence. Curr. Opin. Immunol. 10, 41–44 (1998).

    Article  CAS  PubMed Central  Google Scholar 

  6. Fang, F.C. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat. Rev. Microbiol. 2, 820–832 (2004).

    Article  CAS  Google Scholar 

  7. Muller-Eberhard, H.J. Molecular organization and function of the complement system. Annu. Rev. Biochem. 57, 321–347 (1988).

    Article  CAS  Google Scholar 

  8. Medzhitov, R. & Janeway, C. Jr. Innate immune recognition: mechanisms and pathways. Immunol. Rev. 173, 89–97 (2000).

    Article  CAS  Google Scholar 

  9. Iwanaga, S. The molecular basis of innate immunity in the horseshoe crab. Curr. Opin. Immunol. 14, 87–95 (2002).

    Article  CAS  Google Scholar 

  10. Kawano, T., Pinontoan, R., Hosoya, H. & Muto, S. Monoamine-dependent production of reactive oxygen species catalyzed by pseudoperoxidase activity of human hemoglobin. Biosci. Biotechnol. Biochem. 66, 1224–1232 (2002).

    Article  CAS  Google Scholar 

  11. Twenhofel, W.H. & Shrock, R.R. Invertebrate Paleontology (eds. Twenhofel, W.H. & Shrock, R.R.) 406–477 (McGraw-Hill, New York, 1935).

    Google Scholar 

  12. Ding, J.L. et al. Spatial and temporal coordination of expression of immune response genes during Pseudomonas aeruginosa infection of horseshoe crab, Carcinoscorpius rotundicauda. Genes Immun. 6, 557–574 (2005).

    Article  CAS  Google Scholar 

  13. Decker, H., Ryan, M., Jaenicke, E. & Terwilliger, N. SDS-induced phenol-oxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum, and Cancer magister. J. Biol. Chem. 276, 17796–17799 (2001).

    Article  CAS  Google Scholar 

  14. Bolton, J.L., Trush, M.A., Penning, T.M., Dryhurst, G. & Monks, T.J. Role of quinones in toxicology. Chem. Res. Toxicol. 13, 135–160 (2000).

    Article  CAS  Google Scholar 

  15. Nagai, T. & Kawabata, S. A link between blood coagulation and prophenol oxidase activation in arthropod host defense. J. Biol. Chem. 275, 29264–29267 (2000).

    Article  CAS  Google Scholar 

  16. Nagai, T., Osaki, T. & Kawabata, S. Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides. J. Biol. Chem. 276, 27166–27170 (2001).

    Article  CAS  Google Scholar 

  17. Cerenius, L. & Soderhall, K. The prophenoloxidase-activating system in invertebrates. Immunol. Rev. 198, 116–126 (2004).

    Article  CAS  Google Scholar 

  18. Alayash, A.I. Hemoglobin-based blood substitutes: oxygen carriers, pressor agents, or oxidants? Nat. Biotechnol. 17, 545–549 (1999).

    Article  CAS  Google Scholar 

  19. Fee, J.A. in Oxygen and Oxyradicals in Chemistry and Biology (eds. Rodgers, M.A.J. & Powers, E.L.) 205–239 (Academic, New York, 1981).

    Google Scholar 

  20. Wentworth, A.D., Jones, L.H., Wentworth, P., Janda, K.D. & Lerner, R.A. Antibodies have the intrinsic capacity to destroy antigens. Proc. Natl. Acad. Sci. USA 97, 10930–10935 (2000).

    Article  CAS  Google Scholar 

  21. Bunn, H.F. & Forget, B.G. Hemoglobin: Molecular Genetic and Clinical Aspects (eds. Bunn, H.F. & Forget, B.G.) 634–662 (W.B. Saunders, Philadelphia, 1986).

    Google Scholar 

  22. Skaar, E.P., Humayun, M., Bae, T., DeBord, K.L. & Schneewind, O. Iron-source preference of Staphylococcus aureus infections. Science 305, 1626–1628 (2004).

    Article  CAS  Google Scholar 

  23. Heck, L.W. et al. Degradation of IgA proteins by Pseudomonas aeruginosa elastase. J. Immunol. 144, 2253–2257 (1990).

    CAS  PubMed  Google Scholar 

  24. Wretlind, B. & Pavlovskis, O.R. Pseudomonas aeruginosa elastase and its role in pseudomonas infections. Rev. Infect. Dis. 5, 998–1004 (1983).

    Article  Google Scholar 

  25. Chan, P.F. & Foster, S.J. Role of SarA in virulence determination production and environmental signal transduction in Staphylococcus aureus. J. Bacteriol. 180, 6232–6241 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Zhu, Y., Thangamani, S., Ho, B. & Ding, J.L. The ancient origin of the complement system. EMBO J. 24, 382–394 (2005).

    Article  CAS  Google Scholar 

  27. Ng, P.M., Jin, Z., Tan, S.S., Ho, B. & Ding, J.L. C-reactive protein: a predominant LPS-binding acute phase protein responsive to pseudomonas infection. J. Endotoxin Res. 10, 163–174 (2004).

    Article  CAS  Google Scholar 

  28. Decker, H. & Tuczek, F. Tyrosinase/catecholoxidase activity of hemocyanins: structural basis and molecular mechanism. Trends Biochem. Sci. 25, 392–397 (2000).

    Article  CAS  Google Scholar 

  29. Toder, D.S., Ferrell, S.J., Nezezon, J.L., Rust, L. & Iglewski, B.H. lasA and lasB genes of Pseudomonas aeruginosa: analysis of transcription and gene product activity. Infect. Immun. 62, 1320–1327 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Nellaiappan, K. & Sugumaran, M. On the presence of prophenoloxidase in the hemolymph of the horseshoe crab, Limulus. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 113, 163–168 (1996).

    Article  CAS  Google Scholar 

  31. Lee, S.Y., Lee, B.L. & Soderhall, K. Processing of an antibacterial peptide from hemocyanin of the freshwater crayfish Pacifastacus leniusculus. J. Biol. Chem. 278, 7927–7933 (2003).

    Article  CAS  Google Scholar 

  32. Karlsson, A., Saravia-Otten, P., Tegmark, K., Morfeldt, E. & Arvidson, S. Decreased amounts of cell wall-associated protein A and fibronectin-binding proteins in Staphylococcus aureus sarA mutants due to up-regulation of extracellular proteases. Infect. Immun. 69, 4742–4748 (2001).

    Article  CAS  PubMed Central  Google Scholar 

  33. Dowd, P.F. Relative inhibition of insect phenoloxidase by cyclic fungal metabolites from insect and plant pathogens. Nat. Toxins 7, 337–341 (1999).

    Article  CAS  Google Scholar 

  34. Ariki, S. et al. A serine protease zymogen functions as a pattern-recognition receptor for lipopolysaccharides. Proc. Natl. Acad. Sci. USA 101, 953–958 (2004).

    Article  CAS  Google Scholar 

  35. Zhu, Y., Ng, P.M.L., Wang, L., Ho, B. & Ding, J.L. Diversity in lectins enables immune recognition and differentiation of wide spectrum of pathogens. Int. Immunol. 18, 1671–1680 (2006).

    Article  CAS  Google Scholar 

  36. Kodati, V.R. & Lafleur, M. Comparison between orientational and conformational orders in fluid lipid bilayers. Biophys. J. 64, 163–170 (1993).

    Article  CAS  PubMed Central  Google Scholar 

  37. Osawa, T. & Kato, Y. Protective role of antioxidative food factors in oxidative stress caused by hyperglycemia. Ann. NY Acad. Sci. 1043, 440–451 (2005).

    Article  CAS  Google Scholar 

  38. Hampton, M.B., Kettle, A.J. & Winterbourn, C.C. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 92, 3007–3017 (1998).

    CAS  Google Scholar 

  39. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    Article  CAS  PubMed Central  Google Scholar 

  40. Ding, J.L., Navas, M.A.A. III & Ho, B. Two forms of factor C from the amoebocytes of Carcinoscorpius rotundicauda: purification and characterization. Biochim. Biophys. Acta 1202, 149–156 (1993).

    Article  CAS  Google Scholar 

  41. Wang, J., Ho, B. & Ding, J.L. Functional expression of full length Limulus factor C in stably transformed Sf9 cells. Biotechnol. Lett. 23, 71–76 (2001).

    Article  CAS  Google Scholar 

  42. Brenowitz, M., Bonaventura, C., Bonaventura, J. & Gianazza, E. Subunit composition of high molecular weight oligomer: Limulus polyphemus hemocyanin. Arch. Biochem. Biophys. 210, 748–761 (1981).

    Article  CAS  Google Scholar 

  43. Heinz, D., Margaret, R., Elmar, J. & Nora, T. SDS-induced phenoloxidase activity of hemocyanin from Limulus polyphemus, Eurupelma californicum, and Cancer magister. J. Biol. Chem. 276, 17796–17799 (2001).

    Article  Google Scholar 

  44. Jiang, H., Wang, Y. & Kanost, M.R. Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: a bacteria-inducible protein similar to Drosophila easter. Proc. Natl. Acad. Sci. USA 95, 12220–12225 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B.H. Iglewski (University of Rochester School of Medicine and Dentistry) for P. aeruginosa strains PAO-Iglewski and PAO-B1A1; S. Arvidson (Karolinska University, Sweden) for S. aureus strains PC1839 and AK3; L. Zhang (Institute of Molecular and Cell Biology, Singapore) for plasmid pDSK-GFP; A. Cheung (Dartmouth Medical School) for plasmid pALC1420; H.C. Ng for bacterial isolation and technical support; S. Leptihn for suggestions on bacterial identification by 16S rRNA sequencing; and B. Halliwell, A. Le Saux and P.M.L. Ng for critical reading of the manuscript. Supported by the Agency for Science Technology and Research (Biomedical Research Council), the Academic Research Fund of the Ministry of Education, and the Faculty Research Committee of the National University of Singapore.

Author information

Author notes

  1. Bow Ho and Jeak Ling Ding: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, National University of Singapore, Singapore, 117543

    Naxin Jiang & Jeak Ling Ding

  2. School of Biological Sciences, Nanyang Technological University, Singapore, 637551

    Nguan Soon Tan

  3. Department of Microbiology, National University of Singapore, Singapore, 117597

    Bow Ho

Authors
  1. Naxin Jiang
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Nguan Soon Tan
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Bow Ho
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Jeak Ling Ding
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

N.J. designed and did the experiments and prepared the manuscript; N.S.T. provided some suggestions, helped with real-time fluorescence imaging and manuscript preparation; B.H. provided expertise in microbiology, designed and did some of the experiments and participated in manuscript preparation; J.L.D. conceived the ideas, designed the biochemical, cell and molecular biology experiments, and supervised the studies and preparation of the manuscript.

Corresponding author

Correspondence to Jeak Ling Ding.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Methods and Text (PDF 14224 kb)

Supplementary Video 1

The real-time imaging of the bacterial clearance elicited by PO-catalyzed production of quinone using PAO-Iglewski, the wild type Pseudomonas aeruginosa strain which produces extracellular protease. (MOV 993 kb)

Supplementary Video 2

The real-time imaging of the bacterial clearance elicited by PO-catalyzed production of quinone using PAO-B1A1, the Pseudomonas aeruginosa mutant strain which is protease-deficient. (MOV 1183 kb)

Supplementary Video 3

The real-time imaging of the bacterial clearance elicited by PO-catalyzed production of quinone using PAO-Iglewski in the presence of phenylthiourea, a specific inhibitor of the phenoloxidase. (MOV 803 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jiang, N., Tan, N., Ho, B. et al. Respiratory protein–generated reactive oxygen species as an antimicrobial strategy. Nat Immunol 8, 1114–1122 (2007). https://doi.org/10.1038/ni1501

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni1501

This article is cited by

Search

Advanced 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
pFad - Phonifier reborn

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


Alternative Proxies:

Alternative Proxy

pFad Proxy

pFad v3 Proxy

pFad v4 Proxy