In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

Abstract

The success of Mycobacterium tuberculosis (Mtb) as a human pathogen relies on its ability to resist eradication by the immune system. The identification of mechanisms that enable Mtb to persist is key for finding ways to limit latent tuberculosis, which affects one-third of the world's population. Here we show that conditional gene silencing can be used to determine whether an Mtb gene required for optimal growth in vitro is also important for virulence and, if so, during which phase of an infection it is required. Application of this approach to the prcBA genes, which encode the core of the mycobacterial proteasome, revealed an unpredicted requirement of the core proteasome for the persistence of Mtb during the chronic phase of infection in mice. Proteasome depletion also attenuated Mtb in interferon-γ–deficient mice, pointing to a function of the proteasome beyond defense against the adaptive immune response. Genes that are essential for growth in vitro, in vivo or both account for approximately 20% of Mtb's genome. Conditional gene silencing could therefore facilitate the validation of up to 800 potential Mtb drug targets and improve our understanding of host-pathogen dynamics.

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Figure 1: Tet-ON and Tet-OFF systems allow efficient and rapid silencing of proteasome expression.
Figure 2: In vitro phenotypes caused by prcBA silencing.
Figure 3: Induction of GFP expression in Mtb in mouse lungs.
Figure 4: The proteasome is essential for optimal growth and persistence of Mtb in mice.

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Acknowledgements

We thank G. Lin and C. Nathan (Weill Cornell Medical College) for PrcB-specific antiserum, R. Bryk and C. Nathan (Weill Cornell Medical College) for DlaT-specific antiserum and J. Cox (University of California, San Franciso) for plasmid pJSC284. Wild-type Mtb (H37Rv) was a gift from R. North (Trudeau Institute). We also thank L.M. Pierini for help with fluorescence microscopy, E. Hwang for technical support, C. Nathan and G. Lin for helpful discussions and K.H. Darwin and C. Nathan for reviewing the manuscript. This work was supported by the US National Institutes of Health (grant AI63446 to S.E.), the Irma T. Hirschl Trust (S.E.), the Ellison Medical Foundation (D.S.), the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 473 (W.H.), the Fonds der Chemischen Industrie (W.H.) and the Bill and Melinda Gates Foundation and the Wellcome Trust through the Grand Challenges in Global Health Initiative (S.E., D.S.). The Department of Microbiology and Immunology acknowledges the support of the William Randolph Hearst Foundation.

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S.G. performed experiments. D.S. guided experimental design and analyses. M.M. helped with initial experiments. W.H. provided constructs and advice. S.E. helped with experiments and guided the study. S.G., D.S. and S.E. wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Sabine Ehrt.

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Gandotra, S., Schnappinger, D., Monteleone, M. et al. In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice. Nat Med 13, 1515–1520 (2007). https://doi.org/10.1038/nm1683

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