Sorting Out Antibiotics' Mechanisms of Action: a Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors

Abstract

e In order to accelerate drug discovery, a simple, reliable, and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double-reporter system for not only antimicrobial activity detection but also simultaneous sorting of potential antimicrobials into those that cause ribosome stalling and those that induce the SOS response due to DNA damage. In this reporter system, the red fluorescent protein gene rfp was placed under the control of the SOS-inducible sulA promoter. The gene of the far-red fluorescent protein, katushka2S, was inserted downstream of the tryptophan attenuator in which two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to ribosome-stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need of enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals.T he spread of antibiotic resistance genes among pathogenic bacteria is leading to a gradual decrease in the efficiency of known antibiotics. Substantial efforts have been invested in platforms for new antibiotic development (1). High-throughput screening (HTS) is a major method for the discovery of new chemical scaffolds for drug discovery. However, in the search for new antibiotics, HTS demonstrated low efficiency (for a discussion, see references 2 and 3). Acceleration of the antibiotic development pipeline demands increased efficiency in the identification of mechanisms of action with both HTS of chemical libraries and screening of natural compounds. Ideally, the mechanism of action should be determined while screening for antibacterial activity. One of the major challenges in high-throughput screening is the development of a cost-effective procedure that could maximize information output while concomitantly minimizing the number of pipetting steps and the reagent costs.The most efficient way to reveal the mechanism of action is the application of reporter strains (for a recent review, see reference 4). However, the majority of the reporter strains developed thus far aim to identify a narrow group of chemically related compounds, such as tetracyclines (5), macrolides (6, 7), or ␤-lactams (8). Broader-spectrum reporters based on stress response promoters are also available (9-11). A combination of several reporter strains could help to classify more mechanisms of action, but that would require multiple experiments for a single substance being tested.The majority of antibiotics currently in clinical use target the cell wall, DNA, or protein biosynthesis. For the latter two mechanisms ...

Exploitation of Potentially New Antibiotics from Mangrove Actinobacteria in Maowei Sea by Combination of Multiple Discovery Strategies

“…To accelerate the discovery of antibiotics and to early identify the potential antibacterial mechanism of 83 selected strains, a double fluorescent protein reporter system (pDualrep2) [48] was implemented. The E. coil cell transformed by the plasmid pDualrep2 is a sensor for translation inhibitors, which can be monitored by induction of Katushka2S expression, and DNA damaging agents, monitored by induction of RFP expression.…”

Phazolicin – a Novel Thiazole/Oxazole-Modified Peptide Inhibiting the Bacterial Ribosome in a Species-Specific Way

“…To determine the mode of action of phazolicin we tested its activity in vivo using an E. colibased double-reporter system that allows one to identify antibiotics inducing ribosome stalling or inhibiting DNA replication 19 is an inhibitor of translation (Figure 2A, left panel). In this assay, we observed a difference in the size of PHZ-induced inhibition zones between the E. coli strain with the deleted tolC gene and the wild-type strain (Figure 2A, right panel).…”

“…BW25113ΔtolC-pDualrep2 was used as previously described 19 . Briefly, 2 µL of solutions of tested compounds were applied onto the agar plate that already contained a lawn of the reporter strain.…”

Overview of 17,856 Compound Screening for Translation Inhibition and DNA Damage in Bacteria

“…This promoter is regulated by LexA transcriptional repressor, which is inactivated upon induction of the DNA damage SOS response [19]. The cells transformed by the resulting plasmid pDualrep2 [15] became a sensor for translation inhibitors which might be monitored by induction of Katushka2S expression and DNA damaging agents, such as topoisomerase II inhibitors, monitored by induction of RFP expression. These proteins possess readily distinguishable spectral properties that allow their separate detection via fluorescence scanner.…”

“…On the right is the situation when LexA is inactivated when DNA is damaged. In this case RFP is upregulated lincomycin, clindamycin, nalidixic acid, levofloxacin, ciprofloxacin, tobramycin, neomycin, etoposide, furagin, microcin B17, spectinomycin, etamycin A, hygromycin B, griseoviridin, tylosin, amicoumacin A, fusidic acid, puromycin, and gentamicin were tested against the created reporter strain [15]. It appeared that induction of RFP reporter is caused by all known topoisomerase inhibitors and in addition DNA precursor biosynthesis inhibitors.…”