Fig 1.
Two early stage MEP pathway inhibitors and their targets are shown in the context of the E. coli branchpoint metabolite, DXP.
Butylacetylphosphonate (BAP) is an inhibitor of DXP synthase, and fosmidomycin is an inhibitor of IspC, the first committed step in isoprenoid biosynthesis. (Pi = PO42−).
Fig 2.
E. coli cultures at an initial inoculum density of 106 CFU/mL in M9-glucose were incubated in the presence of BAP () at 4 × MIC (80 μM, or 15 μg/mL), compared to control in the absence of BAP (■) in biological triplicate. Enumeration of bacteria on agar plates over time (0, 2, 4, 6, and 20 h) indicates that BAP is bacteriostatic. (n = 3, error bars represent standard error).
Fig 3.
E. coli treated with 250 μM (47 μg/mL) or 1250 μM (233 μg/mL) BAP in CAMHB (■) or M9-glucose () medium. Intracellular BAP accumulation was monitored by LC-MS (SRM method). BAP uptake is robust and dose-dependent in M9-glucose medium, and poor in CAMHB medium. (n = 3, error bars are standard error, p-values were calculated using an unpaired, 2-sample t-test).
Fig 4.
Checkerboard analysis to assess drug interaction between BAP and fosmidomycin.
a) Representative heat plot showing a synergistic relationship between BAP (5400 μM, 1000 μg/mL) and fosmidomycin (64 μM, 12 μg/mL) in CAMHB growth medium (figure reproduced with permission, [23]). b) Representative heat plot showing an indifferent relationship between BAP (5 μM, 1 μg/mL) and fosmidomycin (340 μM, 62 μg/mL) in M9-glucose minimal medium with an FIC index range of 1–1.25. The most extreme FIC index value is reported above each heat plot.
Table 1.
Minimum inhibitory concentration (MIC) of fosmidomycin against pathogenic bacteria grown in CAMHB or M9-glucose minimal medium.
Fig 5.
Fosmidomycin accumulation in E. coli.
E. coli was treated with 110 μM (20 μg/mL) or 550 μM (100 μg/mL) fosmidomycin in CAMHB (■) or M9-glucose () medium. Intracellular fosmidomycin accumulation was monitored by LC-MS (SRM method). Fosmidomycin uptake is robust and dose-dependent in CAMHB medium, and poor in M9-glucose medium. (n = 3, error bars are standard error, p-values above charts were calculated using an unpaired, 2-sample t-test).
Fig 6.
Fosmidomycin antibacterial activity in GlpT deficient E. coli.
GlpT transporter-containing (■ BW25113) and deficient ( ΔglpT BW25113) E. coli strains treated with 2800 μM (512 μg/mL) fosmidomycin in CAMHB (a), M9-glucose (b), and M9-glycerol (c) growth medium. Cell growth was assessed at 16 h (CAMHB, M9-glucose) or 40 h (M9-glycerol) to ensure culture saturation. The large MIC shift between the parent and GlpT deficient strain in CAMHB and M9-glycerol media indicate that GlpT is a fosmidomycin transporter in these growth conditions. (n = 3, error bars represent standard error).