GWAS and functional studies suggest a role for altered DNA repair in the evolution of drug resistance in Mycobacterium tuberculosis
- National Institute of Immunology, India
- Centre for Cellular and Molecular Biology, India
- Department of Zoology, University of Delhi, India
- Centre for Genetic Manipulation of Crop Plants, University of Delhi South Campus, India
- Department of Microbiology and Cell Biology, Indian Institute of Science Bangalore, India
Figures
Figure 1 with 1 supplement
Genome-wide association study unveils mutations in the DNA repair genes.
(a) Geographical distribution of 2773 clinical strains of Mycobacterium tuberculosis (Mtb). The donut plot represents the proportion of susceptible and drug-resistant (DR) strains in each lineage. …
Figure 1—figure supplement 1
Country-wide distribution of clinical strains.
Each donut plot represents proportion of clinical strains used for the genome-wide association study. Susceptible refers to the antibiotic sensitive strains. DR refers to ‘drug resistance’ to first- …
Figure 2 with 6 supplements
Drug-resistant strains carry mutations in the DNA repair genes.
(a) Volcano plot represents the effect of identified single nucleotide polymorphisms (SNPs) on the development of multidrug resistant/extensively drug resistant TB (MDR/XDR-TB). The positive effect …
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Figure 2—source data 1
Mutations identified in genes that belong to different categories.
- https://cdn.elifesciences.org/articles/75860/elife-75860-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Genome-wide association study analysis.
Bar graph representing the (a) heterozygosity of strains, (b) heterozygosity of markers, and (c) frequency and accumulative frequency of marker density.
Figure 2—figure supplement 2
Linkage disequilibrium, minor allele frequency and quantile-quantile plot in the genome-wide association analysis.
(a) Linkage disequilibrium decay over distance. (b) Minor allele frequency (MAF). (c) Quantile-quantile – plot of p-values.
Figure 2—figure supplement 3
Density of markers in the genome-wide association analysis.
(a–d) Density of markers.
Figure 2—figure supplement 4
Plots showing optimum compression and Type-I error.
(a) The profile for the optimum compression. (b) Type-I error plot.
Figure 2—figure supplement 5
Genome-wide association study-based hypothesis.
In the natural process of evolution, host-imposed stress and antibiotic treatment result in the evolution of wild-type bacteria to multidrug resistant/extensively drug resistant (MDR/XDR). However, …
Figure 2—figure supplement 6
Distribution of mutations in the DNA repair genes.
(a–d) Distribution plot showing single nucleotide polymorphisms (SNPs) in mutY, recF, uvrA, and uvrB identified in drug-resistant strains. Wild type and the alternative alleles are shown. (e–h) …
Figure 3 with 2 supplements
Variants identified in DNA repair genes abrogate their function.
(a) A schematic representation of the base excision repair pathway that operates in mycobacteria. Oxidative damage can result in the conversion of G to 8-oxo-G. If MutM (Fpg) does not repair 8-oxo-G …
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Figure 3—source data 1
Mutation rate analysis in the presence of different drugs.
- https://cdn.elifesciences.org/articles/75860/elife-75860-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Genome-wide association study of lineage 4 strains identified mutations in the DNA repair genes.
(a) Manhattan plot showing identifying genes that belong to DNA repair and direct target of antibiotics. (b) Schematic depicting the generation of gene replacement mutant of mutY. The hygromycinr …
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Figure 3—figure supplement 1—source data 1
Confirmation of gene repalcement mutant and complementation strains.
- https://cdn.elifesciences.org/articles/75860/elife-75860-fig3-figsupp1-data1-v3.zip
Figure 3—figure supplement 2
Mutation frequency analysis.
(a) Mutation frequency was calculated for different strains in the presence of rifampicin (a) or isoniazid (b). (c) Table showing the fold increase in the mutation frequency in comparison with …
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Figure 3—figure supplement 2—source data 1
Analysis of Mutation frequency.
- https://cdn.elifesciences.org/articles/75860/elife-75860-fig3-figsupp2-data1-v3.xlsx
Figure 4
Killing kinetics in the presence of antibiotics show better survival of RvΔmutY and RvΔmutY::mutY R262Q.
(a) Schematic representation of killing kinetics. (b) Growth kinetics in the absence of drugs. (c–f) Growth kinetics in the presence of isoniazid, rifampicin, ciprofloxacin, and ethambutol. Two …
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Figure 4—source data 1
Killing kinetics in the absence and presence of different antibiotics.
- https://cdn.elifesciences.org/articles/75860/elife-75860-fig4-data1-v3.xlsx
Figure 5 with 1 supplement
Mutations in the DNA repair genes provide a survival advantage in the presence of antibiotics.
(a) A schematic is representing the ex vivo infection experiment in the presence and absence of different antibiotics. (b–e) Survival of the strains in the peritoneal macrophages at 4 and 96 hr …
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Figure 5—source data 1
Survival of different strains in the absence and presence of antibiotics ex vivo.
- https://cdn.elifesciences.org/articles/75860/elife-75860-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Ex vivo survival of strains.
(a) Survival of strains with respect to 4 hr post-infection (p.i.) in peritoneal macrophages. (b) CFU enumeration of strains at 4 and 96 hr p.i. (c) Percent survival of each strain in the …
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Figure 5—figure supplement 1—source data 1
Survival of strains before and after passage in the peritoneal macrophages.
- https://cdn.elifesciences.org/articles/75860/elife-75860-fig5-figsupp1-data1-v3.xlsx
Figure 6
WGS reveals the acquisition of direct target mutations in the ex vivo passaged strains.
(a–d) Circos plot showing the WGS analysis of the strains passaged ex vivo in the absence (a) and in the presence of isoniazid (b), rifampicin (c), and ciprofloxacin (d). The outermost circle …
Figure 7
Variant of mutY outcompetes Rv in competition experiment.
(a) Schematic representing the competition experiment performed in peritoneal macrophages. Strains obtained after three rounds of infection in the peritoneal macrophages were used to perform a …
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Figure 7—source data 1
Competition experiment in the presence and absence of different drugs after passage in the peritoneal macrophages.
- https://cdn.elifesciences.org/articles/75860/elife-75860-fig7-data1-v3.xlsx
Figure 8 with 2 supplements
Perturbation of DNA repair results in enhanced survival in vivo.
(a) A schematic representation of the guinea pig infection experiment. (b) Gross histopathology of lungs and spleen of infected guinea pigs. (c) Hematoxylin and eosin staining of infected lung …
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Figure 8—source data 1
Gross histopathology of the infected lungs and spleen isolated from guinea pig.
- https://cdn.elifesciences.org/articles/75860/elife-75860-fig8-data1-v3.pdf
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Figure 8—source data 2
Haematoxylin and eosin staining.
- https://cdn.elifesciences.org/articles/75860/elife-75860-fig8-data2-v3.pdf
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Figure 8—source data 3
Survival of different strains in vivo.
- https://cdn.elifesciences.org/articles/75860/elife-75860-fig8-data3-v3.xlsx
Figure 8—figure supplement 1
Gross histopathology of infected lungs and spleen isolated at 56days post-infection.
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Figure 8—figure supplement 1—source data 1
Gross histopathology of lungs and spleen isolated from guinea pigs after competition experiment.
- https://cdn.elifesciences.org/articles/75860/elife-75860-fig8-figsupp1-data1-v3.pdf
Figure 8—figure supplement 2
WGS analysis of strains isolated from guinea pig lungs.
(a) Circos plot shows the WGS analysis of the strains isolated from guinea pig lungs. The outermost circle represents the reference genome labeled with the known direct target mutations. Circles …
Figure 9
Model.
Model depicts the analysis and subsequent validation. Genome-wide association study (GWAS) revealed mutation in three DNA repair pathway genes in multidrug resistant/extensively drug resistant …
Author response image 1
Author response image 2
Tables
Table 1
Mutations identified in the direct targets of antibiotics.
| Antibiotic | Gene | Mutations identified |
|---|---|---|
| Rifampicin | rpoB | Leu452Pro, Val496Meth |
| Isoniazid | katG | Ser315Thr |
| Ethambutol | embB | Gly406Ser |
| Ofloxacin | gyrA | Ala90Val, Ser91Pro |
| Kanamycin | rrs | 7 independent mutations |
| Pyrazinamide | pncA | His71Arg |
| Ethionamide | ethA | Met95Arg, Pro160(frame-shift) |
| Streptomycin | gidB | Leu35 (frame-shift) |
| Cycloserine | ald | Thr427Pro |
Table 2
Mutations in DNA repair genes associated with drug resistance phenotype.
| Gene | Amino acid change | Wild type | Mutated | False discovery rate-adjusted p-value |
|---|---|---|---|---|
| mutY | Arg262Gln | G | A | 3.83E-09 |
| uvrB | Ala524Val | C | T | 2.15E-07 |
| uvrA | Gln135Lys | C | A | 3.83E-09 |
| RecF | Gly269Gly | G | T | 2.15E-07 |
Table 3
Transporters associated with drug resistance phenotype.
| Gene | False discovery rate-adjusted p-value | Variant |
|---|---|---|
| rv1258c | 1.60E-08 | Non-syn |
| mmpL2 | 9.51E-05 | Non-syn |
| rv0987 | 1.60E-08 | Non-syn |
| rv1250 | 3.44E-07 | Non-syn |
| kdpC | 2.15E-07 | Non-syn |
| rv0928 | 2.15E-07 | Non-syn |
| rv2333c | 1.32E-20 | Frameshift |
| mmpL13a | 1.25E-06 | Syn |
| kdpB | 2.15E-07 | Syn |
Additional files
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Supplementary file 1
Total number of clinical strains used in this study.
The table contains the total number of clinical strains obtained from different studies.
- https://cdn.elifesciences.org/articles/75860/elife-75860-supp1-v3.xlsx
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Supplementary file 2
Clinical strains used for the Genome-wide association analysis.
The table contains the clinical strains which are used for performing genome-wide association study analysis.
- https://cdn.elifesciences.org/articles/75860/elife-75860-supp2-v3.xlsx
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Supplementary file 3
Synonymous change identified in the association analysis.
The table contains synonymous changes identified in the multidrug-resistant/extensively drug-resistant strains.
- https://cdn.elifesciences.org/articles/75860/elife-75860-supp3-v3.xlsx
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Supplementary file 4
Non-synonymous change identified in the association analysis.
The table contains non-synonymous changes identified in the multidrug-resistant/extensively drug-resistant strains.
- https://cdn.elifesciences.org/articles/75860/elife-75860-supp4-v3.xlsx
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Supplementary file 5
Upstream gene variants identified in the association analysis.
The table contains non-upstream gene variants identified in the multidrug-resistant/extensively drug-resistant strains.
- https://cdn.elifesciences.org/articles/75860/elife-75860-supp5-v3.xlsx
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Supplementary file 6
Stop codon or frameshift mutations identified in the association analysis.
The table contains Stop codon or frameshift mutations identified in the multidrug-resistant/extensively drug-resistant strains.
- https://cdn.elifesciences.org/articles/75860/elife-75860-supp6-v3.xlsx
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Supplementary file 7
Codon usage of the multidrug-resistant/extensively drug-resistant (MDR/XDR) strains.
The table contains codon usage in the MDR/XDR and Rv strain.
- https://cdn.elifesciences.org/articles/75860/elife-75860-supp7-v3.xlsx
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Supplementary file 8
Mutations identified in Rv, RvΔmutY, RvΔmutY::mutY, and RvΔmutY::mutY-R262Q strains under different conditions.
The table consists of percentage of mutation and blosum score of genes that harbor s under different conditions.
- https://cdn.elifesciences.org/articles/75860/elife-75860-supp8-v3.xlsx
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Supplementary file 9
Oligonucleotide used in the study.
The table consists of oligonucleotide used in the study.
- https://cdn.elifesciences.org/articles/75860/elife-75860-supp9-v3.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/75860/elife-75860-transrepform1-v3.pdf
