A whole-cell phenotypic screening platform for identifying methylerythritol phosphate pathway-selective inhibitors as novel antibacterial agents

Abstract

Isoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains of Salmonella enterica serovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition.

Fig 1

MEP and MVA pathways for isoprenoid biosynthesis.

Fig 2

Expected screening results for the MEP screening platform. Rows A and B represent an MEP pathway-selective inhibitor and rows C and D an antibacterial that is not selective for the MEP pathway; rows E and F and rows G and H are not antibacterial. Wells A11 and B11 are Fos controls, C11 to H11 are no-inhibitor controls, and column 12 represents media blanks. ● = cell growth; ○ = no cell growth.

Fig 3

Effect of adjusting [IPTG] on CT31-7d growth. Cultures were grown in LB/Kan/Amp with various concentrations of IPTG (serial dilutions starting at 0.02 mM and ending at 0.00039 mM) and no IPTG. Plates were incubated at 37°C and 80% humidity with shaking at 250 rpm for 18 h, at which point A₆₀₀ values were determined. Error bars represent the averages of four independent data points.

Fig 4

Adjusting [IPTG] for CT31-7d growth affects Fos MIC. Cultures were grown in LB/Kan/Amp with various concentrations of Fos (serial dilutions starting at 200 ng/ml and ending at 0.39 ng/ml). Plates were incubated at 37°C and 80% humidity with shaking at 250 rpm for 18 h, at which point A₆₀₀ values were determined. Error bars represent the averages of four independent data points. ■ = 0 mM IPTG, ▲ = 0.002 mM IPTG, ● = 0.02 mM IPTG, ▼ = 0. 2 mM IPTG.

Fig 5

Growth of CT31-7d with or without 100 μg/ml Fos in LB/Kan/Amp and with or without DX and ME supplementation. Black bars = no supplements plus Fos; gray bars = 10 μM DX plus Fos; boxed gray bars = 25 μg/ml ME plus Fos; boxed white bars = 5 mM MVA plus 0.04% ara plus Fos. Cultures were grown in LB/Kan/Amp with the indicated supplements at 37°C and 80% humidity with shaking at 250 rpm for 18 h, at which point A₆₀₀ values were determined. Values represent the means ± standard errors of duplicate data points from two independent experiments.

Fig 6

Schematic of feeding experiments with a DXR inhibitor.