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.

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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.

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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.

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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.

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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

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