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Evolution of high-level ethambutol-resistant tuberculosis through interacting mutations in decaprenylphosphoryl-β-D-arabinose biosynthetic and utilization pathway genes

Nature Genetics volume 45, pages 1190–1197 (2013)Cite this article

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Abstract

To study the evolution of drug resistance, we genetically and biochemically characterized Mycobacterium tuberculosis strains selected in vitro for ethambutol resistance. Mutations in decaprenylphosphoryl-β-D-arabinose (DPA) biosynthetic and utilization pathway genes Rv3806c, Rv3792, embB and embC accumulated to produce a wide range of ethambutol minimal inhibitory concentrations (MICs) that depended on mutation type and number. Rv3806c mutations increased DPA synthesis, causing MICs to double from 2 to 4 μg/ml in a wild-type background and to increase from 16 to 32 μg/ml in an embB codon 306 mutant background. Synonymous mutations in Rv3792 increased the expression of downstream embC, an ethambutol target, resulting in MICs of 8 μg/ml. Multistep selection was required for high-level resistance. Mutations in embC or very high embC expression were observed at the highest resistance level. In clinical isolates, Rv3806c mutations were associated with high-level resistance and had multiplicative effects with embB mutations on MICs. Ethambutol resistance is acquired through the acquisition of mutations that interact in complex ways to produce a range of MICs, from those falling below breakpoint values to ones representing high-level resistance.

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Figure 1: Frequencies of spontaneous resistance to ethambutol.
Figure 2: Mutant strains and MICs generated during stepwise selection for ethambutol resistance.
Figure 3: Relative competitive reductions in fitness for isogenic embB, Rv3806c and/or Rv3792 mutants and strain 4C31-16C1-24C1.
Figure 4: Role of Rv3806c in ethambutol resistance.
Figure 5: Effect of Rv3792 synonymous mutations on Rv3792 and embC mRNA levels.

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Acknowledgements

We thank S. Kuppasani for performing the direct Sanger sequencing. This work was supported in part by National Institute of Allergy and Infectious Diseases, US National Institutes of Health grants AI080653, AI065663 and AI037139 and by Pathogen Functional Genomics Resource Center contract N01-AI5447.

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Author notes

  1. Scott N Peterson & Robert Fleischmann

    Present address: Present addresses: Inflammatory and Infectious Disease Center, Sanford Burnham Medical Research Institute, La Jolla, California, USA (S.N.P.) and National Science Foundation, Arlington, Virginia, USA (R.F.).,

Authors and Affiliations

  1. Division of Infectious Disease, New Jersey Medical School, Rutgers University, Newark, New Jersey, USA

    Hassan Safi, Subramanya Lingaraju & David Alland

  2. Center for Emerging and Re-emerging Pathogens, New Jersey Medical School, Rutgers University, Newark, New Jersey, USA

    Hassan Safi, Subramanya Lingaraju & David Alland

  3. Department of Medicine, New Jersey Medical School, Rutgers University, Newark, New Jersey, USA

    Hassan Safi, Subramanya Lingaraju & David Alland

  4. Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA

    Anita Amin, Michael McNeil & Delphi Chatterjee

  5. Department of Preventive Medicine and Community Health, New Jersey Medical School, Rutgers University, Newark, New Jersey, USA

    Soyeon Kim

  6. Pathogen Functional Genomics Center, J. Craig Venter Institute, Rockville, Maryland, USA

    Marcus Jones, Michael Holmes, Scott N Peterson & Robert Fleischmann

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Contributions

H.S., S.L., A.A., M.J., M.M., S.N.P., D.C., R.F. and D.A. designed experiments. H.S., S.L., A.A. and M.J. performed experiments. H.S., A.A., M.H., M.M., S.N.P., D.C., R.F. and D.A. performed analysis of results. S.K., D.A. and H.S. performed statistical analysis. H.S., S.K., M.M., D.C., R.F. and D.A. wrote the manuscript.

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Correspondence to David Alland.

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Safi, H., Lingaraju, S., Amin, A. et al. Evolution of high-level ethambutol-resistant tuberculosis through interacting mutations in decaprenylphosphoryl-β-D-arabinose biosynthetic and utilization pathway genes. Nat Genet 45, 1190–1197 (2013). https://doi.org/10.1038/ng.2743

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