Identification of cellular targets of a series of boron heterocycles using TIPA II-A sensitive target identification platform.
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.
Department of Biological Sciences, California State University Los Angeles, Los Angeles, CA 90032, USA.
Matthew S
MS
Ward
Department of Biological Sciences, California State University Los Angeles, Los Angeles, CA 90032, USA.
Isba
I
Silva
Department of Biological Sciences, California State University Los Angeles, Los Angeles, CA 90032, USA.
Walfre
W
Martinez
Department of Biological Sciences, California State University Los Angeles, Los Angeles, CA 90032, USA.
Jameka
J
Jefferson
Department of Biological Sciences, California State University Los Angeles, Los Angeles, CA 90032, USA.
Shakila
S
Rahman
Department of Biological Sciences, California State University Los Angeles, Los Angeles, CA 90032, USA.
Jeanie M
JM
Garcia
Department of Chemistry and Biochemistry, California State University East Bay, Hayward, CA 94542, USA.
Divya
D
Kanichar
Department of Chemistry and Biochemistry, California State University East Bay, Hayward, CA 94542, USA.
Lance
L
Roppiyakuda
Department of Chemistry and Biochemistry, California State University East Bay, Hayward, CA 94542, USA.
Ewa
E
Kosmowska
Department of Chemistry and Biochemistry, California State University East Bay, Hayward, CA 94542, USA.
Michelle A
MA
Faust
Department of Chemistry and Biochemistry, California State University East Bay, Hayward, CA 94542, USA.
Kim P
KP
Tran
Department of Chemistry and Biochemistry, California State University East Bay, Hayward, CA 94542, USA.
Felicia
F
Chow
Department of Chemistry and Biochemistry, California State University East Bay, Hayward, CA 94542, USA.
Elena
E
Buglo
Department of Chemistry and Biochemistry, California State University Los Angeles, Los Angeles, CA 90032, USA.
Feimeng
F
Zhou
Department of Chemistry and Biochemistry, California State University East Bay, Hayward, CA 94542, USA.
Michael P
MP
Groziak
Department of Biological Sciences, California State University Los Angeles, Los Angeles, CA 90032, USA. Electronic address: hxu3@calstatela.edu.
H Howard
HH
Xu
D001896:Boron Compounds / Q000737:chemistry* / Q000494:pharmacology; D016503:Drug Delivery Systems / Q000379:methods; D004926:Escherichia coli / Q000187:drug effects; D006571:Heterocyclic Compounds / Q000737:chemistry* / Q000494:pharmacology
D001896:Boron Compounds; D006571:Heterocyclic Compounds
AS19; Boron heterocycles; Essential gene; Inhibitor target identification; Over-expression; Resistance; TIPA