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.

A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis

Abstract

Acidification of the phagosome is considered to be a major mechanism used by macrophages against bacteria, including Mycobacterium tuberculosis (Mtb). Mtb blocks phagosome acidification1, but interferon-γ (IFN-γ) restores acidification and confers antimycobacterial activity2,3. Nonetheless, it remains unclear whether acid kills Mtb, whether the intrabacterial pH of any pathogen falls when it is in the phagosome and whether acid resistance is required for mycobacterial virulence. In vitro at pH 4.5, Mtb survived in a simple buffer and maintained intrabacterial pH. Therefore, Mtb resists phagolysosomal concentrations of acid. Mtb also maintained its intrabacterial pH and survived when phagocytosed by IFN-γ–activated macrophages. We used transposon mutagenesis to identify genes responsible for Mtb's acid resistance. A strain disrupted in Rv3671c, a previously uncharacterized gene encoding a membrane-associated protein, was sensitive to acid and failed to maintain intrabacterial pH in acid in vitro and in activated macrophages. Growth of the mutant was also severely attenuated in mice. Thus, Mtb is able to resist acid, owing in large part to Rv3671c, and this resistance is essential for virulence. Disruption of Mtb's acid resistance and intrabacterial pH maintenance systems is an attractive target for chemotherapy.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Survival of wild-type Mtb and transposon mutants at pH 4.5.
Figure 2: pHIB measurements of acid-sensitive mutants and complementation of the Rv3671c mutant.
Figure 3: The Rv3671c mutant fails to maintain pHIB and is killed within activated macrophages.
Figure 4: Rv3671c is required for Mtb growth and persistence in vivo.

References

  1. MacMicking, J.D., Taylor, G.A. & McKinney, J.D. Immune control of tuberculosis by IFN-γ–inducible LRG-47. Science 302, 654–659 (2003).

    Article  CAS  Google Scholar 

  2. Schaible, U.E., Sturgill-Koszycki, S., Schlesinger, P.H. & Russell, D.G. Cytokine activation leads to acidification and increases maturation of Mycobacterium avium–containing phagosomes in murine macrophages. J. Immunol. 160, 1290–1296 (1998).

    CAS  PubMed  Google Scholar 

  3. Via, L.E. et al. Effects of cytokines on mycobacterial phagosome maturation. J. Cell Sci. 111, 897–905 (1998).

    CAS  PubMed  Google Scholar 

  4. Metchnikoff, E. Immunity to Infective Disease (Cambridge University Press, Cambridge, London, New York, 1905).

    Google Scholar 

  5. Huynh, K.K. & Grinstein, S. Regulation of vacuolar pH and its modulation by some microbial species. Microbiol. Mol. Biol. Rev. 71, 452–462 (2007).

    Article  CAS  Google Scholar 

  6. Ohkuma, S. & Poole, B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc. Natl. Acad. Sci. USA 75, 3327–3331 (1978).

    Article  CAS  Google Scholar 

  7. Armstrong, J.A. & Hart, P.D. Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J. Exp. Med. 134, 713–740 (1971).

    Article  CAS  Google Scholar 

  8. Sturgill-Koszycki, S. et al. Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263, 678–681 (1994).

    Article  CAS  Google Scholar 

  9. Clemens, D.L. & Horwitz, M.A. Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J. Exp. Med. 181, 257–270 (1995).

    Article  CAS  Google Scholar 

  10. Sibley, L.D., Franzblau, S.G. & Krahenbuhl, J.L. Intracellular fate of Mycobacterium leprae in normal and activated mouse macrophages. Infect. Immun. 55, 680–685 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Nathan, C.F., Murray, H.W., Wiebe, M.E. & Rubin, B.Y. Identification of interferon-γ as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158, 670–689 (1983).

    Article  CAS  Google Scholar 

  12. Nathan, C.F. et al. Local and systemic effects of intradermal recombinant interferon-γ in patients with lepromatous leprosy. N. Engl. J. Med. 315, 6–15 (1986).

    Article  CAS  Google Scholar 

  13. Cooper, A.M. et al. Disseminated tuberculosis in interferon-γ gene-disrupted mice. J. Exp. Med. 178, 2243–2247 (1993).

    Article  CAS  Google Scholar 

  14. Flynn, J.L. et al. An essential role for interferon-γ in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178, 2249–2254 (1993).

    Article  CAS  Google Scholar 

  15. Dorman, S.E. et al. Clinical features of dominant and recessive interferon-γ receptor 1 deficiencies. Lancet 364, 2113–2121 (2004).

    Article  CAS  Google Scholar 

  16. Ehrt, S. et al. Reprogramming of the macrophage transcriptome in response to interferon-γ and Mycobacterium tuberculosis: signaling roles of nitric oxide synthase-2 and phagocyte oxidase. J. Exp. Med. 194, 1123–1140 (2001).

    Article  CAS  Google Scholar 

  17. Xie, Q.W. et al. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256, 225–228 (1992).

    Article  CAS  Google Scholar 

  18. MacMicking, J.D. et al. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc. Natl. Acad. Sci. USA 94, 5243–5248 (1997).

    Article  CAS  Google Scholar 

  19. Armstrong, J.A. & Hart, P.D. Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations on bacterial survival. J. Exp. Med. 142, 1–16 (1975).

    Article  CAS  Google Scholar 

  20. Gomes, M.S. et al. Survival of Mycobacterium avium and Mycobacterium tuberculosis in acidified vacuoles of murine macrophages. Infect. Immun. 67, 3199–3206 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. MacGurn, J.A. & Cox, J.S. A genetic screen for Mycobacterium tuberculosis mutants defective for phagosome maturation arrest identifies components of the ESX-1 secretion system. Infect. Immun. 75, 2668–2678 (2007).

    Article  CAS  Google Scholar 

  22. Pethe, K. et al. Isolation of Mycobacterium tuberculosis mutants defective in the arrest of phagosome maturation. Proc. Natl. Acad. Sci. USA 101, 13642–13647 (2004).

    Article  CAS  Google Scholar 

  23. Stewart, G.R., Patel, J., Robertson, B.D., Rae, A. & Young, D.B. Mycobacterial mutants with defective control of phagosomal acidification. PLoS Pathog. 1, 269–278 (2005).

    Article  CAS  Google Scholar 

  24. Dubos, R. The effect of lipids and serum albumin on bacterial growth. J. Exp. Med. 85, 9–22 (1947).

    Article  CAS  Google Scholar 

  25. Kanai, K. & Kondo, E. Antibacterial and cytotoxic aspects of long-chain fatty acids as cell surface events: selected topics. Jpn. J. Med. Sci. Biol. 32, 135–174 (1979).

    Article  CAS  Google Scholar 

  26. Vandal, O.H., Gelb, M.H., Ehrt, S. & Nathan, C.F. Cytosolic phospholipase A2 enzymes are not required by mouse bone marrow–derived macrophages for the control of Mycobacterium tuberculosis in vitro. Infect. Immun. 74, 1751–1756 (2006).

    Article  CAS  Google Scholar 

  27. Ortalo-Magne, A. et al. Identification of the surface-exposed lipids on the cell envelopes of Mycobacterium tuberculosis and other mycobacterial species. J. Bacteriol. 178, 456–461 (1996).

    Article  CAS  Google Scholar 

  28. Darwin, K.H., Ehrt, S., Gutierrez-Ramos, J.C., Weich, N. & Nathan, C.F. The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science 302, 1963–1966 (2003).

    Article  CAS  Google Scholar 

  29. Cole, S.T. et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544 (1998).

    Article  CAS  Google Scholar 

  30. El Ghachi, M., Bouhss, A., Blanot, D. & Mengin-Lecreulx, D. The bacA gene of Escherichia coli encodes an undecaprenyl pyrophosphate phosphatase activity. J. Biol. Chem. 279, 30106–30113 (2004).

    Article  CAS  Google Scholar 

  31. Rose, L., Kaufmann, S.H. & Daugelat, S. Involvement of Mycobacterium smegmatis undecaprenyl phosphokinase in biofilm and smegma formation. Microbes Infect. 6, 965–971 (2004).

    Article  Google Scholar 

  32. Rawlings, N.D., Morton, F.R. & Barrett, A.J. MEROPS: the peptidase database. Nucleic Acids Res. 34, D270–D272 (2006).

    Article  CAS  Google Scholar 

  33. Miesenbock, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    Article  CAS  Google Scholar 

  34. Hart, P.D. & Young, M.R. Ammonium chloride, an inhibitor of phagosome-lysosome fusion in macrophages, concurrently induces phagosome-endosome fusion, and opens a novel pathway: studies of a pathogenic mycobacterium and a nonpathogenic yeast. J. Exp. Med. 174, 881–889 (1991).

    Article  CAS  Google Scholar 

  35. North, R.J. & Jung, Y.J. Immunity to tuberculosis. Annu. Rev. Immunol. 22, 599–623 (2004).

    Article  CAS  Google Scholar 

  36. Mohamedmohaideen, N.N. et al. Structure and function of the virulence-associated high-temperature requirement A of Mycobacterium tuberculosis. Biochemistry 47, 6092–6102 (2008).

    Article  CAS  Google Scholar 

  37. Rohde, K.H., Abramovitch, R.B. & Russell, D.G. Mycobacterium tuberculosis invasion of macrophages: linking bacterial gene expression to environmental cues. Cell Host Microbe 2, 352–364 (2007).

    Article  CAS  Google Scholar 

  38. Stuehr, D.J. & Nathan, C.F. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med. 169, 1543–1555 (1989).

    Article  CAS  Google Scholar 

  39. Nathan, C. Antibiotics at the crossroads. Nature 431, 899–902 (2004).

    Article  CAS  Google Scholar 

  40. Ehrt, S. et al. Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor. Nucleic Acids Res. 33, e21 (2005).

    Article  Google Scholar 

Download references

Acknowledgements

We thank G. Miesenböck (Oxford University) for providing ratiometric pH-GFP; F. Maxfield, J. Roberts and T. Odaira for guidance and support; and L. Cohen-Gould and T. Labissiere at the Electron Microscopy and Histology Core Facilities at Weill Cornell Medical College and Hospital for Special Surgery for assistance with electron microscopy. This work is supported by the US National Institutes of Health (grant PO1 AI056293 to C.F.N.) and the I.T. Hirschl Trust (S.E.). The Department of Microbiology and Immunology acknowledges the support of the William Randolph Hearst Foundation.

Author information

Authors and Affiliations

Authors

Contributions

O.H.V. designed and performed experiments. L.M.P. guided fluorescent microscopy experiments, including their analysis. D.S. guided experimental design and provided constructs. C.F.N. and S.E. guided the study. O.H.V., C.F.N. and S.E. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Carl F Nathan or Sabine Ehrt.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–9 and Supplementary Table 1 (PDF 3199 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Vandal, O., Pierini, L., Schnappinger, D. et al. A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis. Nat Med 14, 849–854 (2008). https://doi.org/10.1038/nm.1795

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.1795

This article is cited by

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