An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

Abstract

With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Overexpression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17-fold and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats.

Bacterial cytological profiling rapidly identifies the cellular pathways targeted by antibacterial molecules

“…BCP has many advantages over other approaches: it is faster, provides higher resolution for identifying more pathways, and can be performed in very small (microliter scale) culture volumes. BCP also does not require a set of specialized strains like many other methods (13)(14)(15)(16)(17). Because a single BCP experiment is sufficient to identify the cellular pathway targeted, the BCP workflow is simple and can be performed in high throughput.…”

“…To overcome the shortcomings inherent in MMS assays, several alternative methods for determining MOA have been developed (8), including isolating resistant mutants, transcriptional profiling, using a collection of strains that are sensitized to hundreds of pathways, or using species with different resistance properties (11)(12)(13)(14)(15)(16)(17). Each of these approaches is complementary to each other and has unique advantages.…”

Challenges of Antibacterial Discovery

“…Recently, a variety of methods based on the use of arrays (transcriptional [121], translational [23], hypersensitization [96], overexpression [394], stress response [24], and synergy/antagonism [397] arrays) have been described that may be useful in identifying the antibacterial mechanism of action of an inhibitor. These methods have also been used for evaluation of leads from enzyme inhibitor programs or with activities discovered through phenotypic or empirical whole-cell screening.…”

Dual Targeting Antibacterial Peptide Inhibitor of Early Lipid A Biosynthesis

“…Overexpression of an essential enzyme to suppress the toxicity of an antibacterial agent is a validated strategy used to identify subcellular targets (23-25). This multicopy suppression strategy was adopted to determine whether or not the antibacterial activity displayed by peptides identified in this study were due to inhibition of LpxD in vivo.…”