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.

  • Letter
  • Published:

The Mycobacterium tuberculosis protein LdtMt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin

Nature Medicine volume 16, pages 466–469 (2010)Cite this article

Subjects

Abstract

The peptidoglycan layer is a vital component of the bacterial cell wall. The existing paradigm describes the peptidoglycan network as a static structure that is cross-linked predominantly by 4→3 transpeptide linkages. However, the nonclassical 3→3 linkages predominate the transpeptide networking of the peptidoglycan layer of nonreplicating Mycobacterium tuberculosis1,2. The molecular basis of these linkages and their role in the physiology of the peptidoglycan layer, virulence and susceptibility of M. tuberculosis to drugs remain undefined. Here we identify MT2594 as an L,D-transpeptidase that generates 3→3 linkages in M. tuberculosis. We show that the loss of this protein leads to altered colony morphology, loss of virulence and increased susceptibility to amoxicillin-clavulanate during the chronic phase of infection. This suggests that 3→3 cross-linking is vital to the physiology of the peptidoglycan layer. Although a functional homolog exists, expression of ldtMt2 is dominant throughout the growth phases of M. tuberculosis. 4→3 transpeptide linkages are targeted by one of the most widely used classes of antibacterial drugs in human clinical use today, β-lactams. Recently, meropenem-clavulanate was shown to be effective against drug-resistant M. tuberculosis3. Our study suggests that a combination of L,D-transpeptidase and β-lactamase inhibitors could effectively target persisting bacilli during the chronic phase of tuberculosis.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Morphology and growth in vitro.
Figure 2: Characterization of LdtMt2 from M. tuberculosis.
Figure 3: Assessment of growth, virulence and susceptibility to amoxicillin in vivo.
Figure 4: Proposed model for physiology of the peptidoglycan layer in M. tuberculosis.

Similar content being viewed by others

References

  1. Wietzerbin, J. et al. Occurrence of D-alanyl-(D)-meso-diaminopimelic acid and meso-diaminopimelyl-meso-diaminopimelic acid interpeptide linkages in the peptidoglycan of Mycobacteria. Biochemistry 13, 3471–3476 (1974).

    Article  CAS  Google Scholar 

  2. Lavollay, M. et al. The peptidoglycan of stationary-phase Mycobacterium tuberculosis predominantly contains cross-links generated by L,D-transpeptidation. J. Bacteriol. 190, 4360–4366 (2008).

    Article  CAS  Google Scholar 

  3. Hugonnet, J.E., Tremblay, L.W., Boshoff, H.I., Barry, C.E. III & Blanchard, J.S. Meropenem-clavulanate is effective against extensively drug-resistant Mycobacterium tuberculosis. Science 323, 1215–1218 (2009).

    Article  CAS  Google Scholar 

  4. Fauci, A.S. Multidrug-resistant and extensively drug-resistant tuberculosis: the National Institute of Allergy and Infectious Diseases Research agenda and recommendations for priority research. J. Infect. Dis. 197, 1493–1498 (2008).

    Article  Google Scholar 

  5. Gandhi, N.R. et al. Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet 368, 1575–1580 (2006).

    Article  Google Scholar 

  6. Jindani, A., Dore, C.J. & Mitchison, D.A. Bactericidal and sterilizing activities of antituberculosis drugs during the first 14 days. Am. J. Respir. Crit. Care Med. 167, 1348–1354 (2003).

    Article  Google Scholar 

  7. Wayne, L.G. & Sohaskey, C.D. Nonreplicating persistence of Mycobacterium tuberculosis. Annu. Rev. Microbiol. 55, 139–163 (2001).

    Article  CAS  Google Scholar 

  8. Betts, J.C., Lukey, P.T., Robb, L.C., McAdam, R.A. & Duncan, K. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol. Microbiol. 43, 717–731 (2002).

    Article  CAS  Google Scholar 

  9. Voskuil, M.I. et al. Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J. Exp. Med. 198, 705–713 (2003).

    Article  CAS  Google Scholar 

  10. Keren, I., Shah, D., Spoering, A., Kaldalu, N. & Lewis, K. Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J. Bacteriol. 186, 8172–8180 (2004).

    Article  CAS  Google Scholar 

  11. Goren, M.B. & Brennan, P.J. Tuberculosis (ed. Youmans, G.P.) 63 (W. B. Saunders, Philadelphia, 1979).

  12. Vollmer, W. & Holtje, J.V. The architecture of the murein (peptidoglycan) in Gram-negative bacteria: vertical scaffold or horizontal layer(s)? J. Bacteriol. 186, 5978–5987 (2004).

    Article  CAS  Google Scholar 

  13. Matsuhashi, M. [Biosynthesis in the bacterial cell wall] Tanpakushitsu Kakusan Koso 11, 875–886 (1966).

    CAS  PubMed  Google Scholar 

  14. Lamichhane, G. et al. A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: application to Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 100, 7213–7218 (2003).

    Article  CAS  Google Scholar 

  15. Mainardi, J.L. et al. A novel peptidoglycan cross-linking enzyme for a β-lactam–resistant transpeptidation pathway. J. Biol. Chem. 280, 38146–38152 (2005).

    Article  CAS  Google Scholar 

  16. Lavollay, M. et al. The β-lactam-sensitive D,D-carboxypeptidase activity of Pbp4 controls the L,D and D,D transpeptidation pathways in Corynebacterium jeikeium. Mol. Microbiol. (in the press) (2009).

  17. Hugonnet, J.E. & Blanchard, J.S. Irreversible inhibition of the Mycobacterium tuberculosis β-lactamase by clavulanate. Biochemistry 46, 11998–12004 (2007).

    Article  CAS  Google Scholar 

  18. Donald, P.R. et al. Early bactericidal activity of amoxicillin in combination with clavulanic acid in patients with sputum smear-positive pulmonary tuberculosis. Scand. J. Infect. Dis. 33, 466–469 (2001).

    Article  CAS  Google Scholar 

  19. Nadler, J.P., Berger, J., Nord, J.A., Cofsky, R. & Saxena, M. Amoxicillin–clavulanic acid for treating drug-resistant Mycobacterium tuberculosis. Chest 99, 1025–1026 (1991).

    Article  CAS  Google Scholar 

  20. Ghuysen, J.M. Serine β-lactamases and penicillin-binding proteins. Annu. Rev. Microbiol. 45, 37–67 (1991).

    Article  CAS  Google Scholar 

  21. Waxman, D.J. & Strominger, J.L. Penicillin-binding proteins and the mechanism of action of β-lactam antibiotics. Annu. Rev. Biochem. 52, 825–869 (1983).

    Article  CAS  Google Scholar 

  22. Crick, D.C. & Brennan, P.J. Biosynthesis of the arabinogalactan-peptidoglycan complex. in The Mycobacterial Cell Envelope (eds. Daffe, M. & Reyrat, J.) 25–40 (American Society for Microbiology, Washington, DC, 2008).

  23. Templin, M.F., Ursinus, A. & Holtje, J.V. A defect in cell wall recycling triggers autolysis during the stationary growth phase of Escherichia coli. EMBO J. 18, 4108–4117 (1999).

    Article  CAS  Google Scholar 

  24. Boneca, I.G. et al. A critical role for peptidoglycan N-deacetylation in Listeria evasion from the host innate immune system. Proc. Natl. Acad. Sci. USA 104, 997–1002 (2007).

    Article  CAS  Google Scholar 

  25. Lee, M.H., Pascopella, L., Jacobs, W.R. Jr. & Hatfull, G.F. Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis and bacille Calmette-Guerin. Proc. Natl. Acad. Sci. USA 88, 3111–3115 (1991).

    Article  CAS  Google Scholar 

  26. Amrein, K.E. et al. Purification and characterization of recombinant human p50csk protein-tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL. Proc. Natl. Acad. Sci. USA 92, 1048–1052 (1995).

    Article  CAS  Google Scholar 

  27. Auger, G., van Heijenoort, J., Mengin-Lecreulx, D. & Blanot, D.A. MurG assay which utilises a synthetic analogue of lipid I. FEMS Microbiol. Lett. 219, 115–119 (2003).

    Article  CAS  Google Scholar 

  28. Arbeloa, A. et al. Synthesis of mosaic peptidoglycan cross-bridges by hybrid peptidoglycan assembly pathways in Gram-positive bacteria. J. Biol. Chem. 279, 41546–41556 (2004).

    Article  CAS  Google Scholar 

  29. Wiegand, I., Hilpert, K. & Hancock, R.E. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3, 163–175 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge the support of US National Institutes of Health award AI30036. This work was also supported by the Foundation pour la Recherche Médicale (Equipe FRM 2006 (Dequation 200661107918)). M. Lavollay is the recipient of an Institut National de la Santé et de la Recherche Médicale PhD fellowship (Poste d'Accueil pour Pharmacien, Médecin, et Vétérinaire).

Author information

Authors and Affiliations

  1. Department of Medicine, Johns Hopkins University, Baltimore, Maryland, USA

    Radhika Gupta, William R Bishai & Gyanu Lamichhane

  2. Centre de Recherche des Cordeliers, Laboratorie de Recherché Moleculaire sur les Antibiotiques, Université Pierre et Marie Curie, Paris, France

    Marie Lavollay, Jean-Luc Mainardi & Michel Arthur

  3. Université Paris Descartes, Paris, France

    Marie Lavollay, Jean-Luc Mainardi & Michel Arthur

  4. Institut National de la Santé et de la Recherche Médicale, Paris, France

    Marie Lavollay, Jean-Luc Mainardi & Michel Arthur

  5. Hôpital Européen Georges Pompidou, Paris, France

    Jean-Luc Mainardi

Authors
  1. Radhika Gupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marie Lavollay
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jean-Luc Mainardi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michel Arthur
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. William R Bishai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gyanu Lamichhane
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.G., W.R.B. and G.L. designed the project. J.-L.M. and M.A. designed the biochemical characterization of MT2594. M.L. and J.-L.M. performed biochemistry and analyzed data. R.G. and G.L. conducted genetics, microbiology and mouse experiments. G.L. wrote the manuscript with contributions from the other authors.

Corresponding author

Correspondence to Gyanu Lamichhane.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–5 and Supplementary Methods (PDF 839 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gupta, R., Lavollay, M., Mainardi, JL. et al. The Mycobacterium tuberculosis protein LdtMt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin. Nat Med 16, 466–469 (2010). https://doi.org/10.1038/nm.2120

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.2120

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