Identification of cellular targets of a series of boron heterocycles using TIPA II-A sensitive target identification platform

Abstract

One of the hurdles in the discovery of antibiotics is the difficulty of linking antibacterial compounds to their cellular targets. Our laboratory has employed a genome-wide approach of over-expressing essential genes in order to identify cellular targets of antibacterial inhibitors. Our objective in this project was to develop and validate a more sensitive disk diffusion based platform of target identification (Target Identification Platform for Antibacterials version 2; TIPA II) using a collection of cell clones in an Escherichia coli mutant (AS19) host with increased outer membrane permeability. Five known antibiotics/inhibitors and 28 boron heterocycles were tested by TIPA II assay, in conjunction with the original assay TIPA. The TIPA II was more sensitive than TIPA because eight boron heterocycles previously found to be inactive to AG1 cells in TIPA assays exhibited activity to AS19 cells. For 15 boron heterocycles, resistant colonies were observed within the zones of inhibition only on the inducing plates in TIPA II assays. DNA sequencing confirmed that resistant clones harbor plasmids with fabI gene as insert, indicating that these boron heterocycles all target enoyl ACP reductase. Additionally, cell-based assays and dose response curved obtained indicated that for two boron heterocycle inhibitors, the fabI cell clone in AG1 (wild-type) host cells exhibited at least 11 fold more resistance under induced conditions than under non-induced conditions. Moreover, TIPA II also identified cellular targets of known antibacterial inhibitors triclosan, phosphomycin, trimethoprim, diazaborine and thiolactomycin, further validating the utility of the new system.

Keywords: AS19; Boron heterocycles; Essential gene; Inhibitor target identification; Over-expression; Resistance; TIPA.

Figure 1

Images of zones of inhibition and resistance colonies (right panels) there within on TIPA II assay trays with filter disks containing either triclosan (top panels) or phosphomycin (bottom panels). Triclosan, 10 μg/disk; Phosphomycin, 200 μg/disk.

Figure 2

(A) Images of zones of inhibition and resistance growth occurred within the zone of inhibition (right) on TIPA II induced assay tray containing Pool #5 with filter disks applied with 1.1 mg of trimethoprim. (B) Dose responses and resistance testing for clone JW0047 containing plasmid-borne folA gene (AS19 host) against trimethoprim under induced (blue) or non-induced (red) conditions.

Figure 3

Comparison of TIPA (AG1 as host cells) and TIPA II (AS19 as host cells). Images of zones of inhibition (or lack of) and resistance colonies within the zone of inhibition on TIPA II induced assay trays with filter disks containing either 37 or 44. For TIPA assays, each disk was loaded with 750 μg. For TIPA II assays, 4 μg/disk for 37, and 15 μg/disk for 44 were used.

Figure 4

Images of zones of inhibition on TIPA assay trays containing Pool #2 with filter disks soaked with the following compounds: a, 31; b, 24. Each disk was loaded with 75 μg of compound.

Figure 5

Dose responses and resistance testing for clone JW1281 (AG1 host) against 31 (A) and 24 (B) under non-induced (0 mM IPTG) and induced (1 mM IPTG) conditions. The AG1 cell clone JW1281 harbors a plasmid containing the fabl gene whose expression is inducible by IPTG.

Figure 6

Images of zones of inhibition on assay trays with Pool #2 focusing on filter disks containing thiolactomycin (300 μg/disk) without induction (left) or with induction (right). Left panel shows complete clearing within zone of inhibition illustrating growth inhibition. Right panel shows emergence of resistant colonies within the zone of inhibition.