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.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Corbett, E.L. et al. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch. Intern. Med. 163, 1009–1021 (2003).
Gomez, J.E. & McKinney, J.D. M. tuberculosis persistence, latency, and drug tolerance. Tuberculosis (Edinb.) 84, 29–44 (2004).
Sassetti, C.M., Boyd, D.H. & Rubin, E.J. Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol. 48, 77–84 (2003).
Sassetti, C.M. & Rubin, E.J. Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. USA 100, 12989–12994 (2003).
Ehrt, S. et al. Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor. Nucleic Acids Res. 33, e21 (2005).
Ehrt, S. & Schnappinger, D. Controlling gene expression in mycobacteria. Future Microbiol. 1, 177–184 (2006).
Guo, X.V. et al. Silencing essential protein secretion in Mycobacterium smegmatis using tetracycline repressors. J. Bacteriol. 189, 4614–4623 (2007).
Chalut, C., Botella, L., de Sousa-D'Auria, C., Houssin, C. & Guilhot, C. The nonredundant roles of two 4′-phosphopantetheinyl transferases in vital processes of Mycobacteria. Proc. Natl. Acad. Sci. USA 103, 8511–8516 (2006).
Hu, G. et al. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate. Mol. Microbiol. 59, 1417–1428 (2006).
Lin, G. et al. Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity. Mol. Microbiol. 59, 1405–1416 (2006).
De Mot, R., Nagy, I., Walz, J. & Baumeister, W. Proteasomes and other self-compartmentalizing proteases in prokaryotes. Trends Microbiol. 7, 88–92 (1999).
Knipfer, N. & Shrader, T.E. Inactivation of the 20S proteasome in Mycobacterium smegmatis. Mol. Microbiol. 25, 375–383 (1997).
Nagy, I. et al. Characterization of a novel intracellular endopeptidase of the α/β hydrolase family from Streptomyces coelicolor A3(2). J. Bacteriol. 185, 496–503 (2003).
Hong, B. et al. Inactivation of the 20S proteasome in Streptomyces lividans and its influence on the production of heterologous proteins. Microbiology 151, 3137–3145 (2005).
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).
Lamichhane, G. et al. Deletion of a Mycobacterium tuberculosis proteasomal ATPase homologue gene produces a slow-growing strain that persists in host tissues. J. Infect. Dis. 194, 1233–1240 (2006).
Darwin, K.H., Lin, G., Chen, Z., Li, H. & Nathan, C.F. Characterization of a Mycobacterium tuberculosis proteasomal ATPase homologue. Mol. Microbiol. 55, 561–571 (2005).
Pickart, C.M. & Cohen, R.E. Proteasomes and their kin: proteases in the machine age. Nat. Rev. Mol. Cell Biol. 5, 177–187 (2004).
Festa, R.A., Pearce, M.J. & Darwin, K.H. Characterization of the proteasome accessory factor (paf) operon in Mycobacterium tuberculosis. J. Bacteriol. 189, 3044–3050 (2007).
Berens, C. & Hillen, W. Gene regulation by tetracyclines. Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes. Eur. J. Biochem. 270, 3109–3121 (2003).
Ji, Y. et al. Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA. Science 293, 2266–2269 (2001).
Lathem, W.W., Price, P.A., Miller, V.L. & Goldman, W.E. A plasminogen-activating protease specifically controls the development of primary pneumonic plague. Science 315, 509–513 (2007).
North, R.J. & Jung, Y.J. Immunity to tuberculosis. Annu. Rev. Immunol. 22, 599–623 (2004).
Flynn, J.L. et al. An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178, 2249–2254 (1993).
De Mot, R., Schoofs, G. & Nagy, I. Proteome analysis of Streptomyces coelicolor mutants affected in the proteasome system reveals changes in stress-responsive proteins. Arch. Microbiol. 188, 257–271 (2007).
Grune, T. et al. Peroxynitrite increases the degradation of aconitase and other cellular proteins by proteasome. J. Biol. Chem. 273, 10857–10862 (1998).
Munoz-Elias, E.J. et al. Replication dynamics of Mycobacterium tuberculosis in chronically infected mice. Infect. Immun. 73, 546–551 (2005).
Demartino, G.N. & Gillette, T.G. Proteasomes: machines for all reasons. Cell 129, 659–662 (2007).
Duncan, K. & Barry, C.E., III Prospects for new antitubercular drugs. Curr. Opin. Microbiol. 7, 460–465 (2004).
Bardarov, S. et al. Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 148, 3007–3017 (2002).
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.
About this article
Cite this article
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
PLOS Pathogens (2021)
Applied Sciences (2020)
Translational Research (2020)