Article Figures & Data
Figures
- FIG 1
Identification of a small molecule that is antimicrobial to S. aureus. (A) Workflow for identifying compounds of interest. (B) Percent growth inhibition by treatment with the indicated concentrations of compound ‘921 relative to vehicle control for S. aureus Newman or ΔmntH/C strain. Data are mean ± standard deviation from duplicate measurements. Statistical significance was determined by t test where * = P < 0.05 and ** = P < 0.01. (C) Relative growth at 12 h with 5 μM ‘921 ± MnCl2 is graphed as % of growth without compound or MnCl2. Data are mean ± standard deviation from triplicate measurements acquired on two separate days and combined (n = 6). Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test comparing each concentration of Mn to no-Mn control where *** = P < 0.001. (D) CFU recovered at 2-h intervals from growth curve of S. aureus Newman treated with vehicle, 5 μM ‘921, or 5 μM ‘921 + 1 mM MnCl2 over time. Data are mean ± standard deviation from quadruplicate measurements. (E) CFU recovered following 24-h treatment of mid-exponential-phase S. aureus Newman cultures with the indicated combinations of vehicle, 1 mM MnCl2, 100 μM ‘921, and 1 mM EDTA. Data are mean ± standard deviation from triplicate measurements acquired on two separate days combined (n = 6). Statistical significance was determined by one-way ANOVA with Sidak’s multiple-comparison test comparing each concentration of Mn to no-Mn control where * = P < 0.05 and *** = P < 0.001. Experiments in panels F and G were performed in an anaerobic chamber. (F) Growth of S. aureus Newman in the presence of vehicle, 1 mM MnCl2, 5 μM ‘921, or 5 μM ‘921 + 1 mM MnCl2. Data are mean ± standard deviation for triplicate measurements. (G) CFU recovered following treatment of mid-exponential-phase cultures of S. aureus with vehicle, 1 mM or 100 μM MnCl2, 100 μM ‘921, or combinations of MnCl2 + ‘921 for 24 h. Data are mean ± standard deviation for triplicate measurements.
- FIG 2
VU0026921 induces a metal toxicity transcriptional response. RNA sequencing was performed on mid-exponential S. aureus Newman cultures exposed to vehicle, 1 mM MnCl2, 100 μM ‘921, or 100 μM ‘921 + 1 mM MnCl2 for 30 min. (A) Multidimensional scaling visualization of RNA sequencing biological replicates. The x and y axes are dimensionless units. The closeness of symbols to each other on the 2-dimensional plot represents the relatedness of the transcriptional profiles of the represented data sets. (B) Categories of genes whose transcription was significantly different between 100 μM ‘921 treatment alone and cotreatment with 1 mM MnCl2. (C) Fold change (log2) of transcript abundance for all S. aureus genes following treatment with VU0026921 compared to vehicle-treated controls. (D) Fold change (log2) of transcript abundance for all S. aureus genes following treatment with VU0026921 and 1 mM MnCl2 compared to ‘921 alone. For panels C and D, genes involved in metal homeostasis and reactive oxygen species (ROS) detoxification are highlighted. Dashed lines indicate genes with greater than 2-fold change and a false-discovery rate of less than 0.01.
- FIG 3
VU0026921 causes metal accumulation and oxidative stress in S. aureus. Metal isotopes 55Mn (A), 59Co (B), 63Cu (C), 56Fe (D), and 66Zn (E) were measured by ICP-MS in S. aureus Newman treated for 30 min with vehicle, 1 mM MnCl2, 100 μM ‘921, or 100 μM ‘921 + 1 mM MnCl2. Data are mean ± standard deviation normalized to 34S to account for differences in growth of four biological replicates. Statistical significance was determined by one-way ANOVA where ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and **** = P < 0.0001. (F) ROS levels in S. aureus cultures treated with vehicle, 50 μM ‘921, 1 mM MnCl2, or 50 μM ‘921 + 1 mM MnCl2 for 6 h. Data are mean ± standard deviation for six biological replicates. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test where ** = P < 0.01 and **** = P < 0.0001. (G) CFU recovered following 24-h treatment of mid-exponential-phase cultures of S. aureus Newman, ΔmntH/C, or ΔsodA/sodM with vehicle, 1 mM MnCl2, 100 μM ‘921, or 1 mM Mn + 100 μM ‘921. Data are mean ± standard deviation combined from three independent triplicate experiments, performed on separate days (n = 9). Statistical significance was determined by one-way ANOVA with Dunnett’s multiple-comparison test where ### = P < 0.001 compared to WT vehicle-treated bacteria and ** = P < 0.01 and *** = P < 0.001 for the comparisons indicated by the bars. (H) S. aureus Newman was treated with vehicle, 50 μM ‘921, 80 μM MitoTEMPO, or 50 μM ‘921 + 80 μM MitoTEMPO, and growth was monitored by optical density at 600 nm for 24 h. Data are mean ± standard deviation for six biological replicates.
- FIG 4
Mn and Co protect S. aureus against VU0026921 toxicity, while Cu exacerbates it. (A to D and F) CFU recovered following 4-h treatment of S. aureus Newman with vehicle, 100 μM ‘921, 100 μM FeSO4 (A), ZnCl2 (B), MnCl2 (C), CoCl2 (D), and CuSO4 (F), or 10 μM ‘921 + 100 μM metal. Data presented are mean ± standard deviation from triplicate measurements. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test where ns = P > 0.05, *** = P < 0.001, and **** = P < 0.0001. (E and G) ROS levels in S. aureus cultures treated with vehicle, 50 μM ‘921, 100 μM CoCl2 (E) or CuSO4 (G), and 50 μM ‘921 + 100 μM metal for 6 h. Data are mean ± standard deviation for six biological replicates. Statistical significance was determined for each condition compared to ‘921 treatment by one-way ANOVA with Tukey’s multiple-comparison test where *** = P < 0.001 and **** = P < 0.0001. (H) USA300, an MRSA strain, and USA300 ΔcopAZ ΔcopBL (Δcop) were treated with vehicle, 5 μM ‘921, 10 μM CuSO4, or 5 μM ‘921 + 10 μM CuSO4, and growth was monitored by optical density at 600 nm for 24 h. Data are mean ± standard deviation for six biological replicates.
- FIG 5
Binding of divalent transition metals stabilized VU0026921. (A to H) Absorbance was measured from 280 nm to 600 nm in 10-nm increments of 0 or 100 μM ‘921 combined with vehicle (A) or 500 μM CoCl2 (B), CuSO4 (C), FeSO4 (D), MnCl2 (E), ZnCl2 (F), CaCl2 (G), or MgCl2 (H). Absorbance measurements were taken at 0, 5, 15, 30, 60, or 120 min after the addition of metals. The final absorbance spectra are ‘921 + metal with spectra for metal alone subtracted. Spectra displayed are representative of a single experiment that was performed three times. The spectrum of ‘921 at 0 min was included in each panel as a reference of the compound absorbance alone.
- FIG 6
Chemical features of VU0026921 required for toxicity. (A) Chemical structures of VU0026921 and analogs. (B) Relative potency of 100 μM (each) analog—VU0026921 (‘921), VU0849731 (‘731), VU0849732 (‘732), VU0849730 (‘730), or VU0849729 (‘729)—measured as growth of S. aureus as a percentage of untreated cells at 4 h for each compound tested at the indicated concentrations. Data are mean ± standard deviation from triplicate measurements. (C) S. aureus Newman was treated with vehicle, 100 μM ‘921, or 100 μM ‘921 analog, and growth was monitored by optical density at 600 nm for 24 h. Data are mean ± standard deviation from triplicate measurements. (D) ROS levels in S. aureus cultures treated with vehicle, 50 μM ‘921, or 50 μM ‘921-2 for 6 h. Data are mean ± standard deviation for six biological replicates. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test where ns = P > 0.05 and **** = P < 0.0001.
- FIG 7
VU0026921 S. aureus killing is not through fatty acid biosynthesis inhibition. (A and B) MIC of irgasan (A), a FabI inhibitor that halts fatty acid biosynthesis and inhibits S. aureus strain RN4220, and VU0026921 (B). Irgasan has an increased MIC in S. aureus RN4220 mutants with fatty acid biosynthesis disabled when cotreated with oleic acid, while ‘921 (B) does not, indicating that ‘921 does not inhibit fatty acid biosynthesis. Note: no growth is observed for accD mutants in the absence of oleic acid. (C and D) Platensimycin (C), a FabF inhibitor, has an increased MIC in E. faecalis when cotreated with oleic acid, while ‘921 (D) does not, suggesting that ‘921 does not inhibit fatty acid biosynthesis in E. faecalis. All data are mean ± standard deviation for triplicate measurements. Note: 10 μg/ml was the highest concentration of platensimycin tested. The actual MIC of platensimycin against E. faecalis in the presence of oleic acid may be higher.
- FIG 8
VU0026921 is growth inhibitory toward Gram-positive bacteria. (A to F) S. aureus MW2 (A), UAMS1 (B), HG003 (C), 8325-4 (D), RN6390 (E), and SH1000 (F) were treated with vehicle or 5 μM or 20 μM ‘921, and growth was monitored by optical density at 600 nm for 24 h. Data are mean ± standard deviation from triplicate measurements. (G and H) The Gram-positive organisms Bacillus anthracis (G) and Micrococcus luteus (H) were treated with vehicle or 5 μM ‘921, and growth was monitored by optical density at 600 nm for 16 h. Data are mean ± standard deviation from quadruplicate measurements.
Supplemental Material
TABLE S1
(A) MICs for compounds tested for antimicrobial activity against WT and ΔmntH/C S. aureus. MIC was defined as minimum concentration of compound that inhibited growth by at least 50% at 8 h. (B) Mn rescue of compound-mediated growth inhibition at 5 μM. *Rescue: concentration of Mn that resulted in 50% increase in S. aureus growth at 8 h. #N/A indicates that compound is not inhibitory to WT at 5 μM. Download Table S1, XLSX file, 0.1 MB.
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FIG S1
Antimicrobial activity of VU0026921. (A) S. aureus Newman was treated with vehicle, 5 μM ‘921, or 5 μM ‘921 + 1 mM MnCl2, and growth was monitored by optical density at 600 nm for 20 h. Data are mean ± standard deviation from triplicate measurements. (B and C) S. aureus Newman was untreated or treated with 50 μM (B) or 3 μM (C) ‘921 that was incubated with shaking in TSB at 37°C for 0, 4, 8, or 24 h prior to the addition of the bacterial inoculum to assess ‘921 compound stability, and growth was monitored by optical density at 600 nm for 24 h. Data are mean ± standard deviation from triplicate measurements. Download FIG S1, TIF file, 2.7 MB.
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TABLE S2
(A) Transcripts significantly* changed# by treatment with 100 μM ‘921. *Significance was defined as false-discovery rate (FDR) < 0.001. #Defined as |log2 fold change| >2. (B) COG analysis of upregulated and downregulated genes in S. aureus upon VU0026921 treatment. Download Table S2, XLSX file, 0.04 MB.
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TABLE S3
Transcripts significantly* changed# between 100 μM ‘921 and 100 μM ‘921 + 1 mM MnCl2 combination treatment. *Significance was defined as FDR < 0.001. #Defined as >2 log2 fold change. Download Table S3, XLSX file, 0.03 MB.
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TABLE S4
Transcripts significantly* changed# by treatment with 1 mM MnCl2. *Significance was defined as FDR < 0.001. #Defined as >2 log2 fold change. Download Table S4, XLSX file, 0.01 MB.
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TABLE S5
Expression of genes involved in metal homeostasis and detoxification following treatment with VU0026921 with or without Mn. Download Table S5, XLSX file, 0.01 MB.
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FIG S2
EDTA lowers ‘921-induced metal uptake, and Co protects S. aureus against VU0026921 killing. (A to E) Metal isotopes 55Mn (A), 59Co (B), 63Cu (C), 56Fe (D), and 66Zn (E) were measured by ICP-MS in S. aureus Newman treated for 30 min with vehicle, 100 μM EDTA, 100 μM ‘921, or 100 μM ‘921 + 100 μM EDTA. Data are mean ± standard deviation normalized to 34S to account for differences in growth of four biological replicates. Statistical significance was determined by one-way ANOVA where ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and **** = P < 0.0001. (F) S. aureus Newman was treated with vehicle or 25 μM ‘921 and increasing molar ratios of CoCl2 to ’921. At 6 h following treatment, growth was determined by optical density at 600 nm. Data are mean ± standard deviation for six biological replicates. Statistical significance was determined for each condition compared to vehicle treatment by one-way ANOVA with Tukey’s multiple-comparison test where ns = P > 0.05 and **** = P < 0.0001. (G) S. aureus Newman was treated with vehicle, 10 μM ‘921, 100 μM CoCl2 or vitamin B12, or 10 μM ‘921 + 100 μM CoCl2 or vitamin B12, and growth was monitored by optical density at 600 nm for 24 h. Data are mean ± standard deviation for six biological replicates. Download FIG S2, TIF file, 2.7 MB.
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FIG S3
VU0026921 is not growth inhibitory towards Gram-negative bacteria. The Gram-negative organisms E. coli DH5α (A), E. coli SLO1B (B), E. coli MG1655 (C), E. coli BL21 (D), P. aeruginosa PAO1 (E), P. aeruginosa PA14 (F), K. pneumoniae TOP2 (G), and A. baumannii 17978 (H) were treated with vehicle or 0 μM (untreated) or 160 μM ‘921, and growth was monitored by optical density at 600 nm for 24 h. Data are mean ± standard deviation from triplicate measurements. Download FIG S3, TIF file, 2.7 MB.
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TABLE S6
Bacterial strains used in this study. Download Table S6, XLSX file, 0.01 MB.
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TABLE S7
Primers used in this study. Download Table S7, XLSX file, 0.01 MB.
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