A macroevolution-inspired approach to reveal novel antibiotic resistance mechanisms
- Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, United Kingdom
- Proteomics Scientific Technology Platform, The Francis Crick Institute, United Kingdom
- Department of Chemistry, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, United States
Figures
Figure 1
A diverse species library of the genus Mycobacterium.
(a) Phylogenetic tree of mycobacterial species in our library calculated using the bcgTree pipeline v 1.1.0 [62, 65-68]. (b) Doubling time. (c) Genome size. (d) Guanine and cytosine percentage (GC%). (e) Ribosomal copies (rrn operon). (f) Gene ontology (GO) distribution. Colors from left to right represent the following GO categories: regulation of DNA-templated transcription (highlighted with a light orange arrow), transmembrane transport (highlighted with a dark orange arrow); amino acid, lipid, carbohydrate derivative, nucleobase-containing small molecule, and carbohydrate metabolic processes; generation of precursor metabolites and energy; sulfur compound, vitamin, and tRNA metabolic processes; DNA repair, protein modification process, signaling, cell wall organization or biogenesis, cellular modified amino acid metabolic process, DNA replication, DNA recombination, ribosome biogenesis, anatomical structure development, protein catabolic process, protein-containing complex assembly, protein maturation, nitrogen cycle metabolic process, intracellular protein transport, metal ion homeostasis, cell division, protein secretion, mRNA metabolic process, DNA integration, transport, organic substance transport, defense response to other organism, organic substance biosynthetic process, organic substance metabolic process, cellular process, nitrogen compound transport, regulation of gene expression, cellular biosynthetic process, and other metabolic processes.
Figure 2 with 4 supplements
Antibiotic sensitivity mapping reveals complex patterns.
(a) Heatmaps of overall MIC99 values. In the X axis, antibiotics are organized based on their mechanism of action; in the Y axis, mycobacterial species are organized phylogenetically in the left heatmap and based on their response to the set of antibiotics tested (Manhattan clustering) in the right heatmap. Colors represent the standardized MIC99 (mean/SD and centered scaled). Lower MIC99 values are in brown/orange and higher MIC99 values in lilac/purple. The details of the data can be found in Figure 2—figure supplement 2. Radar plots displaying the standardized MIC99 for all antibiotics tested. MIC99 ¬ values are normalized to be plotted in radar plots. All radar plots display the results for M. tuberculosis in orange. Mycobacterium branderi is displayed in purple, Mycobacterium conceptionense in dark blue, and M. smegmatis in light blue. (c) Violin plots showing the distribution of MIC99 values. In the X axis, the set of antibiotics tested; in the Y axis the MIC99 values in µg/mL. (d) Relationship between mycobacterial doubling time (X axis) and MIC99 for the antibiotics BDQ, LZD, and RIF (Y axis). Antibiotics targeting the cell wall are: isoniazid (INH), ethionamide (ETH), ethambutol (EMB), d-cycloserine (DCS), and meropenem (MEM); RNA/protein synthesis: rifampicin (RIF), streptomycin (STR), kanamycin (KAN), amikacin (AMK), capreomycin (CAP), and linezolid (LZD); DNA gyrase: moxifloxacin (MFX) and ofloxacin (OFX); folate metabolism: para-aminosalicylic acid (PAS); and ATP synthase: bedaquiline (BDQ).
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Figure 2—source data 1
Mass spectrometry data used to quantify rifampin in mycobacteria.
- https://cdn.elifesciences.org/articles/101940/elife-101940-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Detailed overall MIC99 quantifications against the mycobacterial species in our library.
X axes represent the species tested in phylogenetic order, and Y axes are the MIC99 measurements in log10 scale. Dotted lines represent the mean MIC99 for each drug.
Figure 2—figure supplement 2
Log2(FC) MIC99 (Y axis) compared to the mean MIC99 for each antibiotic.
Species organized by phylogeny (X axis). Dotted line represents the mean MIC99 for each antibiotic.
Figure 2—figure supplement 3
Detailed view of the Mycobacterium holsaticum group MIC99 values.
Figure 2—figure supplement 4
Primary sequence alignment of Rv2172c-encoded methylene tetrahydrofolate reductase and its homolog from M. holsaticum.
Red arrows indicate residues that lead to decreased enzymatic activity and increased sensitivity to PAS.
Figure 3
Intrabacterial antibiotic concentration does not correlate with potency.
(a) Positive mode extracted ion chromatograms (EICs) of whole-cell extracts of mycobacteria treated with selected antibiotics. BDQ (m/z 555.1642), LZD (m/z 338.1511), and RIF (m/z 823.4124). (b) Chemical structure of BDQ, LZD, and RIF. (c) Relative intracellular antibiotic concentration, obtained by comparing the peak height of the samples with and injection of 10 µM of antibiotic, then normalized by the concentration of antibiotic used to treat the cells. (d) Relative intracellular antibiotic concentrations in relation to the MIC99. Data in (c) and (d) come from independent experiments.
Figure 4 with 1 supplement
High-level rifampicin resistance is caused by rifamycin modification in selected mycobacteria.
(a) RIF MIC99 values for the mycobacterial species in our library organized in decreasing MIC99 value order. (b) Cultures of selected species on solid medium (7H10) containing RIF at different concentrations, starting at 1× M. tuberculosis MIC. (c) Comparison of the amino acid residue sequence of the rifampicin resistance-determining region (RRDR) of RpoB in selected mycobacterial species; the only residue that differs is Ser 450 in M. branderi. (d and e) Volcano plots showing the differential protein expression in whole-cells with and without RIF revealing inducible expression of RIF ADP-ribosyltransferase 1 (Arr-1) in M. smegmatis, M. conceptionense, and M. flavescens. (f) Detection and quantification of ribosyl-RIF in whole-cell extracts by LC-MS.
Figure 4—figure supplement 1
Phylogenetic distribution of RIF ADP-ribosyl transferases present in the mycobacterial species in our library.
(b) Quantification of ribosyl-RIF in some mycobacterial species that encode Arr-1 and/or Arr-X. Not detected (ND).
Figure 5
Characterization of a novel rifabutin-ADP ribosyltransferase in mycobacteria.
(a) Phylogenetic tree of mycobacterial RIF ADP-ribosyltransferases (RIF-ARTs) and related PFAM family PF12120 sequences. Many mycobacterial species encode the equivalent of M. smegmatis RIF-ART (MSMEG_1221; Arr-1/ms in purple), and some mycobacterial species encode a previously unidentified sister group we have named Arr-X (dark orange). See Supplementary file 5 for detailed information of the sequences in the tree. (b) Ribbon representation of the crystal structure of M. smegmatis RIF-ART (PDB code 2HW2; light blue) with RIF bound (red) overlaid with the AlphaFold2 models of M. conceptionense Arr-X (dark blue) and M. flavescens Arr-X (dark orange) (Jumper et al., 2021). (c) Apparent velocity of reaction of each of the enzymes (X axis) with different rifamycins as substrate. (d) M. conceptionense single and double knockdown (KD) arr strains in the presence of rifabutin.
Author response image 1
Author response image 2
Author response image 3
Structure and sequence conservation of RpoB in selected mycobacterial species.
(A) Rainbow diagram of RpoB X-Ray structure coloured according to sequence conservation. Dark purple indicates high conservation, whereas dark orange indicates low conservation. RIF (showed in magenta) is bound to RpoB. Zoomed view displays that the RIF-binding pocket is considerably conserved. (B) RpoB protein sequence has an 81bp region called Rifampicin Resistance Determining Region (RRDR) that is known to be important for RIF binding and is where most mutations occur in drug-resistant TB. Sequence alignment displays that the RRDR region is conserved with the exception of M. branderi, which has an Asn instead of a Ser residue in position 456 (numbering is related to the M. tuberculosis sequence), highlighted in bold.
Additional files
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Supplementary file 1
Information on antibiotics used in this study.
- https://cdn.elifesciences.org/articles/101940/elife-101940-supp1-v1.xlsx
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Supplementary file 2
List of mycobacterial Arr orthologues.
- https://cdn.elifesciences.org/articles/101940/elife-101940-supp2-v1.csv
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Supplementary file 3
Primary sequence variation of Arr 1 and 3.
- https://cdn.elifesciences.org/articles/101940/elife-101940-supp3-v1.xlsx
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Supplementary file 4
MIC for different rifamycins and sgRNA sequences.
- https://cdn.elifesciences.org/articles/101940/elife-101940-supp4-v1.xlsx
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Supplementary file 5
Rifampin ADP-Ribosyltransferase phylogenetic tree calculation and sequence IDs.
- https://cdn.elifesciences.org/articles/101940/elife-101940-supp5-v1.csv
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MDAR checklist
- https://cdn.elifesciences.org/articles/101940/elife-101940-mdarchecklist1-v1.docx
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A macroevolution-inspired approach to reveal novel antibiotic resistance mechanisms
eLife 13:RP101940.
https://doi.org/10.7554/eLife.101940.3
