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:

CARD9 facilitates microbe-elicited production of reactive oxygen species by regulating the LyGDI-Rac1 complex

Nature Immunology volume 10, pages 1208–1214 (2009)Cite this article

Abstract

In response to invading microorganisms, macrophages engage in phagocytosis and rapidly release reactive oxygen species (ROS), which serve an important microbicidal function. However, how phagocytosis induces ROS production remains largely unknown. CARD9, a caspase-recruitment domain (CARD)-containing protein, is important for resistance to fungal and bacterial infection. The mechanism of CARD9-mediated bacterial clearance is still mostly unknown. Here we show that CARD9 is required for killing intracellular bacteria in macrophages. CARD9 associated with the GDP-dissociation inhibitor LyGDI in phagosomes after bacterial and fungal infection and binding of CARD9 suppressed LyGDI-mediated inhibition of the GTPase Rac1, thereby leading to ROS production and bacterial killing in macrophages. Thus, our studies identify a key pathway that leads to microbe-elicited ROS production.

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: Card9−/− macrophages are defective in killing L. monocytogenes.
Figure 2: Genome-wide screening for CARD9-interacting proteins.
Figure 3: Subcellular location of the interaction between CARD9 and LyGDI.
Figure 4: Localization of LyGDI and CARD9 after bacterial infection.
Figure 5: Inducible interaction between CARD9 and LyGDI.
Figure 6: CARD9 regulates Rac1 activation.
Figure 7: Colocalization of Rac1 and p47phox with bacteria-containing phagosomes.
Figure 8: ROS production in BMDMs.

Similar content being viewed by others

References

  1. Mosser, D.M. & Edwards, J.P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8, 958–969 (2008).

    Article  CAS  Google Scholar 

  2. Scott, C.C., Botelho, R.J. & Grinstein, S. Phagosome maturation: a few bugs in the system. J. Membr. Biol. 193, 137–152 (2003).

    Article  CAS  Google Scholar 

  3. Lambeth, J.D. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4, 181–189 (2004).

    Article  CAS  Google Scholar 

  4. Sumimoto, H., Miyano, K. & Takeya, R. Molecular composition and regulation of the Nox family NAD(P)H oxidases. Biochem. Biophys. Res. Commun. 338, 677–686 (2005).

    Article  CAS  Google Scholar 

  5. Groemping, Y. & Rittinger, K. Activation and assembly of the NADPH oxidase: a structural perspective. Biochem. J. 386, 401–416 (2005).

    Article  CAS  Google Scholar 

  6. Bokoch, G.M. & Diebold, B.A. Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood 100, 2692–2696 (2002).

    Article  CAS  Google Scholar 

  7. Heasman, S.J. & Ridley, A.J. Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat. Rev. Mol. Cell Biol. 9, 690–701 (2008).

    Article  CAS  Google Scholar 

  8. Rossman, K.L., Der, C.J. & Sondek, J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat. Rev. Mol. Cell Biol. 6, 167–180 (2005).

    Article  CAS  Google Scholar 

  9. Dovas, A. & Couchman, J.R. RhoGDI: multiple functions in the regulation of Rho family GTPase activities. Biochem. J. 390, 1–9 (2005).

    Article  CAS  Google Scholar 

  10. DerMardirossian, C. & Bokoch, G.M. GDIs: central regulatory molecules in Rho GTPase activation. Trends Cell Biol. 15, 356–363 (2005).

    Article  CAS  Google Scholar 

  11. Bretscher, A., Edwards, K. & Fehon, R.G. ERM proteins and merlin: integrators at the cell cortex. Nat. Rev. Mol. Cell Biol. 3, 586–599 (2002).

    Article  CAS  Google Scholar 

  12. Allenspach, E.J. et al. ERM-dependent movement of CD43 defines a novel protein complex distal to the immunological synapse. Immunity 15, 739–750 (2001).

    Article  CAS  Google Scholar 

  13. Takahashi, K. et al. Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein. J. Biol. Chem. 272, 23371–23375 (1997).

    Article  CAS  Google Scholar 

  14. Janmey, P.A. & Lindberg, U. Cytoskeletal regulation: rich in lipids. Nat. Rev. Mol. Cell Biol. 5, 658–666 (2004).

    Article  CAS  Google Scholar 

  15. Hsu, Y.M. et al. The adaptor protein CARD9 is required for innate immune responses to intracellular pathogens. Nat. Immunol. 8, 198–205 (2007).

    Article  CAS  Google Scholar 

  16. Gross, O. et al. Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity. Nature 442, 651–656 (2006).

    Article  CAS  Google Scholar 

  17. Hara, H. et al. The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors. Nat. Immunol. 8, 619–629 (2007).

    Article  CAS  Google Scholar 

  18. Schnupf, P. & Portnoy, D.A. Listeriolysin O: a phagosome-specific lysin. Microbes Infect. 9, 1176–1187 (2007).

    Article  CAS  Google Scholar 

  19. Hamon, M., Bierne, H. & Cossart, P. Listeria monocytogenes: a multifaceted model. Nat. Rev. Microbiol. 4, 423–434 (2006).

    Article  CAS  Google Scholar 

  20. Myers, J.T., Tsang, A.W. & Swanson, J.A. Localized reactive oxygen and nitrogen intermediates inhibit escape of Listeria monocytogenes from vacuoles in activated macrophages. J. Immunol. 171, 5447–5453 (2003).

    Article  CAS  Google Scholar 

  21. Ding, Z. et al. A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners. Proc. Natl. Acad. Sci. USA 103, 15014–15019 (2006).

    Article  CAS  Google Scholar 

  22. Zal, T. Visualization of protein interactions in living cells. Adv. Exp. Med. Biol. 640, 183–197 (2008).

    Article  CAS  Google Scholar 

  23. Kerppola, T.K. Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu Rev Biophys 37, 465–487 (2008).

    Article  CAS  Google Scholar 

  24. Ciruela, F. Fluorescence-based methods in the study of protein-protein interactions in living cells. Curr. Opin. Biotechnol. 19, 338–343 (2008).

    Article  CAS  Google Scholar 

  25. Liu, D., Yang, X., Yang, D. & Songyang, Z. Genetic screens in mammalian cells by enhanced retroviral mutagens. Oncogene 19, 5964–5972 (2000).

    Article  CAS  Google Scholar 

  26. Scherle, P., Behrens, T. & Staudt, L.M. Ly-GDI, a GDP-dissociation inhibitor of the RhoA GTP-binding protein, is expressed preferentially in lymphocytes. Proc. Natl. Acad. Sci. USA 90, 7568–7572 (1993).

    Article  CAS  Google Scholar 

  27. Lelias, J.M. et al. cDNA cloning of a human mRNA preferentially expressed in hematopoietic cells and with homology to a GDP-dissociation inhibitor for the rho GTP-binding proteins. Proc. Natl. Acad. Sci. USA 90, 1479–1483 (1993).

    Article  CAS  Google Scholar 

  28. Goodridge, H.S. et al. Differential use of CARD9 by Dectin-1 in macrophages and dendritic cells. J. Immunol. 182, 1146–1154 (2009).

    Article  CAS  Google Scholar 

  29. Pick, E., Gorzalczany, Y. & Engel, S. Role of the rac1 p21-GDP-dissociation inhibitor for rho heterodimer in the activation of the superoxide-forming NADPH oxidase of macrophages. Eur. J. Biochem. 217, 441–455 (1993).

    Article  CAS  Google Scholar 

  30. Abo, A. et al. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature 353, 668–670 (1991).

    Article  CAS  Google Scholar 

  31. Benard, V., Bohl, B.P. & Bokoch, G.M. Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J. Biol. Chem. 274, 13198–13204 (1999).

    Article  CAS  Google Scholar 

  32. Bokoch, G.M. Regulation of the phagocyte respiratory burst by small GTP-binding proteins. Trends Cell Biol. 5, 109–113 (1995).

    Article  CAS  Google Scholar 

  33. Shaughnessy, L.M. & Swanson, J.A. The role of the activated macrophage in clearing Listeria monocytogenes infection. Front. Biosci. 12, 2683–2692 (2007).

    Article  CAS  Google Scholar 

  34. Blander, J.M. & Medzhitov, R. On regulation of phagosome maturation and antigen presentation. Nat. Immunol. 7, 1029–1035 (2006).

    Article  CAS  Google Scholar 

  35. Shiloh, M.U. et al. Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. Immunity 10, 29–38 (1999).

    Article  CAS  Google Scholar 

  36. Wells, C.A. et al. The macrophage-inducible C-type lectin, mincle, is an essential component of the innate immune response to Candida albicans. J. Immunol. 180, 7404–7413 (2008).

    Article  CAS  Google Scholar 

  37. Yamasaki, S. et al. Mincle is an ITAM-coupled activating receptor that senses damaged cells. Nat. Immunol. 9, 1179–1188 (2008).

    Article  CAS  Google Scholar 

  38. Sato, K. et al. Dectin-2 is a pattern recognition receptor for fungi that couples with the Fc receptor γ chain to induce innate immune responses. J. Biol. Chem. 281, 38854–38866 (2006).

    Article  CAS  Google Scholar 

  39. Himes, S.R., Cronau, S., Mulford, C. & Hume, D.A. The Runx 1 transcription factor controls CSF-1-dependent and -independent growth and survival of macrophages. Oncogene 24, 5278–5286 (2005).

    Article  CAS  Google Scholar 

  40. Daniel, D.S. et al. The reduced bactericidal function of complement C5-deficient murine macrophages is associated with defects in the synthesis and delivery of reactive oxygen radicals to mycobacterial phagosomes. J. Immunol. 177, 4688–4698 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank W. Li and F. Southwick (University of Florida) for GFP–L. monocytogenes, and X. Qin (University of Texas MD Anderson Cancer Center) for the retroviral packaging vector pCGP and pVSV-G. Supported by the National Institutes of Health (RO1AI050848 and R01GM065899 to X.L. and R01GM069572 to Z.S.), the Welch Foundation (Z.S.) and the Leukemia and Lymphoma Society (X.L. and Z.S.).

Author information

Author notes

  1. Weihui Wu and Yen-Michael S Hsu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Weihui Wu, Yen-Michael S Hsu, Liangkuan Bi & Xin Lin

  2. Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Xin Lin

  3. The Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA

    Zhou Songyang

Authors
  1. Weihui Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yen-Michael S Hsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Liangkuan Bi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhou Songyang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xin Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.W., Y.-M.S.H. and L.B. did the experiments; W.W. and X.L. designed the experiments, analyzed the results and wrote the manuscript; and Z.S. provided the reagents and technical advice.

Corresponding author

Correspondence to Xin Lin.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 626 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, W., Hsu, YM., Bi, L. et al. CARD9 facilitates microbe-elicited production of reactive oxygen species by regulating the LyGDI-Rac1 complex. Nat Immunol 10, 1208–1214 (2009). https://doi.org/10.1038/ni.1788

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.1788

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