A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculosis is critical for prompt diagnosis and optimal treatment, particularly for toxic second-line drugs such as D-cycloserine. Here we used the whole-genome sequences from 498 strains of M. tuberculosis to identify new resistance-conferring genotypes. By combining association and correlated evolution tests with strategies for amplifying signal from rare variants, we found that loss-of-function mutations in ald (Rv2780), encoding L-alanine dehydrogenase, were associated with unexplained drug resistance. Convergent evolution of this loss of function was observed exclusively among multidrug-resistant strains. Drug susceptibility testing established that ald loss of function conferred resistance to D-cycloserine, and susceptibility to the drug was partially restored by complementation of ald. Clinical strains with mutations in ald and alr exhibited increased resistance to D-cycloserine when cultured in vitro. Incorporation of D-cycloserine resistance in novel molecular diagnostics could allow for targeted use of this toxic drug among patients with susceptible infections.
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The authors would like to express their sincere gratitude to the patients in South Africa and China who provided clinical samples, without which this study would not have been possible. The authors also gratefully acknowledge the collaborative efforts of the following individuals whose significant previous contributions allowed for this study: W.R. Bishai, N. Bantubani, L. Alvarado, S.B. Chapman, N.R. Mvelase, E.Y. Duffy, M.G. Fitzgerald, P. Govender, S. Gujja, S. Hamilton, C. Howarth, J.D. Larimer, M.D. Pearson, M.E. Priest, and Q. Zeng. M. bovis ATCC 19210 was generously provided by R. Warren (Stellenbosch University). The authors would also like to thank C. Cuomo and three anonymous reviewers for providing helpful comments on the manuscript. Additionally, the authors would like to acknowledge the Broad Sequencing Platform for assistance with data acquisition.
This project has been funded in part with federal funds from NIAID, NIH, US Department of Health and Human Services, under contract HHSN272200900018C, IeDEA (NIH grant 5U01AI069924-07), and grant U19AI110818 to the Broad Institute. K.A.C. was supported by NHLBI grant T32HL007633. T.A. was supported by a postdoctoral fellowship from the Research Foundation–Flanders. Additional funding sources included grant U19AI51794 and the US Centers for Disease Control and Prevention. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Significance of associations between genotypic variants and drug resistance phenotypes excluding strains with known resistance-conferring genotypes on a per-drug basis.
Each circle represents a genotypic feature that is plotted at the intersection of the negative log-transformed P values from the Fisher’s exact and correlated evolution tests for each drug. P values were corrected for multiple comparisons using the Benjamini–Hochberg method. Genotypic variants known to confer resistance are colored according to the drug to which they confer resistance, and genotypes with no known effect on drug resistance are shown in gray. Genotypes scoring well in both tests appear in the upper right quadrants.
Supplementary Figure 2 A single-gene knockout of ald has a shorter time to positivity than wild-type M. tuberculosis when cultured in the presence of d-cycloserine.
Four laboratory strains—wild-type M. tuberculosis (WT), Δald (ald knockout), ald knockout complemented with ald (Δald-comp), and BCG—were cultured in varying concentrations of d-cycloserine. The color legend indicates the concentration of d-cycloserine in μg/ml. Strains were set up in triplicate, and the resulting time to positivity in MGIT was recorded as days since inoculation. The mean time to positivity is plotted with error bars to represent s.e.m. P values were calculated using two-way ANOVA.
Supplementary Figure 3 Evolution of ald, alr, ddlA, cycA, and pykA across the M. tuberculosis complex (MTBC).
A maximum-likelihood phylogeny was estimated using strains representing the diversity of the MTBC and specifically the vaccine strain BCG. Acquisition of nonsynonymous and loss-of-function mutations in the genes mentioned above was then reconstructed using parsimony and depicted at the appropriate nodes. Clades other than BCG with uniform genotypes were collapsed for visualization. Loss of function of both ald and pykA occurs in conjunction with the loss of region of difference 9 (RD9); pykA loss of function is reverted in both BCG and M. suricattae (indicated above as gain of function, GOF). A cycA loss-of-function mutation also occurs at the base of the M. microti–M. pinnipedii clade. Two mutations were identified in nearly all strains except for the reference H37Rv and close relatives (cycA R93L and ddlA T365A), suggesting that they are phylogenetic mutations and not correlative of resistance to any drugs.
Supplementary Figure 4 Restoration of wild-type ald in BCG inhibits growth in the presence of higher concentrations of d-cycloserine.
As BCG, which is innately resistant to d-cycloserine, contains a frameshift mutation in ald, a wild-type copy of M. tuberculosis ald was used for complementation back into BCG (BCG-ald-comp). Both BCG and BCG-ald-comp were subsequently cultured in varying concentrations of d-cycloserine in MGIT, and the time to positivity was normalized to that for the no-drug control for each strain to calculate the growth inhibition index. P values were calculated using two-way ANOVA.
Supplementary Figure 5 Restoration of wild-type ald in BCG confers longer time to positivity in the presence of higher concentrations of d-cycloserine.
As BCG, which is innately resistant to d-cycloserine, contains a frameshift mutation in ald, a wild-type copy of M. tuberculosis ald was used for complementation back into BCG (BCG-ald-comp). Both BCG and BCG-ald-comp were subsequently cultured in varying concentrations of d-cycloserine in MGIT. Strains were assessed in triplicate, and mean time to positivity in MGIT in days is plotted with the s.e.m. The concentration of d-cycloserine is given in μg/ml. P values were calculated using two-way ANOVA.
Supplementary Figures 1–5, Supplementary Tables 1, 2 and 5–12, and Supplementary Note. (PDF 1731 kb)
Top 20 scoring features from the Fisher's exact and correlated evolution tests for each drug from the analysis of all strains. (XLSX 59 kb)
Top 20 scoring features from the Fisher's exact and correlated evolution tests for each drug from the analysis of strains without known resistance-onferring mutations. (XLSX 59 kb)
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Desjardins, C., Cohen, K., Munsamy, V. et al. Genomic and functional analyses of Mycobacterium tuberculosis strains implicate ald in D-cycloserine resistance. Nat Genet 48, 544–551 (2016). https://doi.org/10.1038/ng.3548
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