Fig 1.
Chemical structures of PYRC (top) and EQI (bottom).
Table 1.
IC90 (μg/mL) and IC50 data for pyrrolocin B, PYRC and EQI.
Fig 2.
Potentiation of EQI treated in combination with polymyxin B.
Percent inhibition of polymyxin B (0.125 μg/mL) and equisetin (32 μg/mL) treated singly and in combination against E. coli (ATCC 23724). Polymyxin B is represented by “polB”.
Fig 3.
PCA of transcriptomic responses of Mtb cultures to anti-TB drug treatments.
Mtb cultures were treated in triplicate with ciproflaxin (red), DMSO (blue), gentamicin (green), isoniazid (purple), para-aminosalicylic acid (yellow), PYRC (cyan), pyrazindamide (brown), or rifampicin (magenta) for 6 hours at 10X IC90 or IC90. Vehicle (DMSO) cluster (circled in blue); transcription synthesis inhibitor cluster (circled in red); protein synthesis inhibitor cluster (circled in green); and PYRC cluster (circled in cyan). RNA-seq was performed and data analysis was completed in Partek®Genomic Suite®.
Fig 4.
Total metal analysis of EQI-treated S. aureus by ICP-OES and metal rescue.
Panel A), metal analysis of equisetin-treated S. aureus S. aureus culture was added to LB broth, with DMSO or EQI (IC90; 4.36 μM). Cultures were incubated at 37°C for 2 hours, digested in nitric acid (trace metal grade) and analyzed by ICP-OES. Total metal concentrations were adjusted based on final OD600. Co, Cd, Ni, Ag, & Se total metal concentrations < Limit of Detection. Mean ± SEM, n = 11. *p<0.05; **p<0.005 (Student’s t-test). Panel B), S. aureus cultures were incubated in DMSO or EQI (IC90; 4.36 μM) in the presence or absence of exogenous metals. Metal solutions (MgCl, MnCl, & Fe(EDTA)) were: 1 μM, 10 μM, and 100 μM. Percent survival determined by the MTT assay. Mean ± SEM, n = 6. *p<0.05 (Student’s t-test).
Table 2.
EQI decreases malonyl-CoA levels in S. aureus.
Fig 5.
Fatty acid supplementation can overcome growth inhibition by PYRC and EQI treatment in S. aureus.
S. aureus was grown in the presence of drug in LB+BSA (No FAs; black circle), LB+BSA plus a15:0/a17:0 (500 μM, 2:1 ratio of a15:0 to a17:0; red triangle), or LB+BSA plus 18:1Δ9 (500 μM; blue square). Mean data of two independent experiment run in triplicate are shown ± standard error of the mean. Significance was determined using a two-tailed, unpaired t test. *, p < 0.05 (No FAs vs a15:0/a17:0): †, p < 0.05 (No FAs vs 18:1Δ9). A) Percent growth of S. aureus in the presence of PYRC. B) Percent growth of S. aureus in the presence of EQI.
Fig 6.
PYRC and EQI impair [1-14C] acetate incorporation in S. aureus.
S. aureus was incubated with [1-14C] acetate (4μCi/mL) for 2 hours in the presence of DMSO, PYR or EQI. A) left panel, representative X-ray film of lipid extract from S. aureus following PYRC treatment eluted on a reverse-phase TLC plate. DMSO control indicated as “D”. The triangle indicates an increase in drug concentration from left to right. Right panel, percent incorporation of [1-14C] acetate following PYRC treatment normalized to DMSO. Mean data of two independent experiments are shown ± standard error of the mean. B) Left panel, representative X-ray film of lipid extract from S. aureus following EQI treatment eluted on a reverse-phase TLC plate. DMSO control indicated as “D”. The triangle indicates an increase in drug concentration from left to right. Right panel, percent incorporation of [1-14C] acetate following EQI treatment normalized to DMSO. Mean data of two independent experiments are shown ± standard error of the mean. C) Representative X-ray film of lipid extract from S. aureus following PYRC and EQI treatment eluted on a normal-phase TLC plate. DMSO control indicated as “D”. The triangle indicates a decrease in drug concentration from left to right. Tested concentrations of EQI: 0.7, 1.5, 3, 5, and 15 μM; PYRC 3.5, 7, 14, 28, and 56 μM. Left panel, percent incorporation of [1-14C] acetate following EQI treatment normalized to DMSO. Right panel, percent incorporation of [1-14C] acetate following PYRC treatment normalized to DMSO.
Fig 7.
Densitometry scan of X-ray film from normal-phase TLC of incorporation of [1-14C] acetate into lipid fraction from S. aureus following PYRC treatment.
Performed as in Fig 6C, right), above, and normalized to DMSO control. A) Densitometry scan of DMSO control lane on X-ray film, increasing Rf, left to right. Spots quantified across PYRC concentrations included Start (origin) and discernable densitometry peaks (spots) 2–7. B) Relative grayscale of film exposure densities at the tested PYRC concentrations, 0, 1.5, 3, 6, 12 and 25 μM. Incorporation of radiolabeled [1-14C] acetate was decreased uniformly across the radiolabeled components of the extract in a dose-related fashion.
Fig 8.
Inhibition of the biotin carboxylase (BC) and carboxyltransferase (CT) components of acetyl-CoA carboxylase by Pyrrolocin C and Equisetin.
The concentration of each inhibitor was 0.1 mM. A. Percent activity of biotin carboxylase in the presence of Pyrrolocin C or Equisetin. The initial velocity of biotin carboxylase in the absence of inhibitor was measured and set to 100%. The substrates biotin (40 mM) and ATP (0.2 mM) were held constant at subsaturating levels. B. Percent activity of carboxyltransferase in the presence of Pyrrolocin C or Equisetin. The initial velocity of carboxyltransferase in the absence of inhibitor was measured and set to 100%. The substrates biocytin (6 mM) and malonyl-CoA (0.04 mM) were held constant at subsaturating levels. All assays were performed in triplicate ± S.D.