Fig. 1 Antibiotic-induced metabolome responses in M. smegmatis.

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Fig. 1 Antibiotic-induced metabolome responses in M. smegmatis. Antibiotic-induced metabolome responses in M. smegmatis. (A) Metabolomics workflow. Cells were grown in 700-μl volumes in 96-well plates to an OD595 of about 0.4, before addition of 10 μl of the antimicrobial compound. Cell culture (80 μl) was withdrawn from each well at each sampling time. Forty microliters was used to determine cell density, and the remaining 40 μl was added to cold extraction buffer. Supernatant was directly injected into a time-of-flight mass spectrometer, and relative changes in metabolite intensities were extrapolated from processing of the metabolome data. (B) Compounds tested. Almost half of the compounds tested included different concentrations of reference antimicrobials (yellow) and chemical stress agents (green) with known MoAs; the remainder were compounds from a GSK library used at 10 μM concentration (blue). (C) Distribution of MoAs for the 62 reference compounds. ATP, adenosine 5′-triphosphate. (D) Schematic representation of the drug-metabolome response data set. For each antimicrobial compound tested, the dynamic profile of 1006 metabolites was interrogated. As an example, the top graph illustrates the response of the folic acid biosynthesis intermediate 4-aminobenzoic acid to the antimicrobial para-aminosalicylic acid (PAS) (red, 104 μM; gray, 41 μM; blue, 25 μM). The bottom graph shows the response of the bacterial metabolite mycobactin to the known antimicrobial isoniazid (red, 1.5 mM; gray, 0.22 mM; blue, 0.11 mM). Thick lines represent the results from the impulse model fitting analysis for the three drug concentrations. Metabolic profiles of 4-aminobenzoic acid and mycobactin across all conditions are shown in light gray. (E) Distribution of metabolic response onset times for antibiotics belonging to the seven main antibiotic categories tested in this study. The onset time is defined as the time at which metabolite changes reached half of their maximum change after treatment of M. smegmatis with the compounds. For each perturbation (treatment with compound), metabolites with a model fitting R2 ≥ 0.6 and a maximum absolute log2 fold change ≥ 2 were retained. Mattia Zampieri et al., Sci Transl Med 2018;10:eaal3973 Published by AAAS