Amplified Detection of Transcriptional and Translational Inhibitors in Bioluminescent Escherichia coli K-12

Galluzzi, Lorenzo; Karp, Matti

doi:10.1177/1087057103008003012

Cited by 11 publications

(9 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Differences in reporter gene expression versus the numbers of CFU have been noted previously, particularly with specific classes of drugs, such as fluoroquinolones and ␤-lactams (19,27). The sensitivity of LORA to fluoroquinolones was enhanced by extending the anaerobic incubation time from 7 to 10 days (data not shown), but while this decreased the MIC obtained by LORA, a discrepancy between luciferase assayand CFU-based MICs remained.…”

Section: Vol 51 2007 Lora For High-throughput Screening Of Compoundsupporting

confidence: 53%

Low-Oxygen-Recovery Assay for High-Throughput Screening of Compounds against Nonreplicating Mycobacterium tuberculosis

Cho

Warit

Wan

et al. 2007

Antimicrob Agents Chemother

286

370

View full text Add to dashboard Cite

Screening for new antimicrobial agents is routinely conducted only against actively replicating bacteria. However, it is now widely accepted that a physiological state of nonreplicating persistence (NRP) is responsible for antimicrobial tolerance in many bacterial infections. In tuberculosis, the key to shortening the 6-month regimen lies in targeting this NRP subpopulation. Therefore, a high-throughput, luminescence-based lowoxygen-recovery assay (LORA) was developed to screen antimicrobial agents against NRP Mycobacterium tuberculosis. M. tuberculosis H 37 Rv containing a plasmid with an acetamidase promoter driving a bacterial luciferase gene was adapted to low oxygen conditions by extended culture in a fermentor with a 0.5 headspace ratio. The MICs of 31 established antimicrobial agents were determined in microplate cultures maintained under anaerobic conditions for 10 days and, for comparative purposes, under aerobic conditions for 7 days. Cultures exposed to drugs under anaerobic conditions followed by 28 h of "recovery" under ambient oxygen produced a luminescent signal that was, for most compounds, proportional to the number of CFU determined prior to the recovery phase. No agents targeting the cell wall were active against NRP M. tuberculosis, whereas drugs hitting other cellular targets had a range of activities. The calculated Z factor was in the range of 0.58 to 0.84, indicating the suitability of the use of LORA for high-throughput assays. This LORA is sufficiently robust for use for primary high-throughput screening of compounds against NRP M. tuberculosis.It is now widely accepted that a physiological state of nonreplicating persistence (NRP) is responsible for antimicrobial tolerance in many bacterial infections (11). In tuberculosis, a subpopulation of Mycobacterium tuberculosis isolates in NRP is considered an important contributing factor to the long treatment duration required, and the key to shortening the currently recommended 6-month regimen (the primary goal of new anti-M. tuberculosis chemotherapy) lies in effective targeting of this phenotype (2, 6). Standard drug susceptibility assays for detection of the activities of drugs against rapidly growing bacteria may not identify such compounds (8,11). In vitro models of M. tuberculosis isolates in NRP exist (4,30,(37)(38)(39), and there are several reports of studies that have assessed drug activity against NRP or stationary-phase cells (20,(34)(35)(36)42); however, these have relied upon the enumeration of CFU, thus precluding high-throughput screening (HTS) applications and requiring a minimum of 3 to 4 weeks for the completion of testing. In addition, the culture and preparation of NRP M. tuberculosis cells (5,17,29,30,38) have used batch cultures, which are not optimal for monitoring and for comparative studies. In contrast, a chemostat or a fermentor with a continuous-oxygen-monitoring culture system allows bacteria to be grown in a controlled and defined environment; there are two reports of the successful cultivation of M. tuberculos...

show abstract

Section: Vol 51 2007 Lora For High-throughput Screening Of Compoundsupporting

confidence: 53%

Low-Oxygen-Recovery Assay for High-Throughput Screening of Compounds against Nonreplicating Mycobacterium tuberculosis

Cho

Warit

Wan

et al. 2007

Antimicrob Agents Chemother

286

370

View full text Add to dashboard Cite

show abstract

“…Such systems were created on the basis of beta-galactosidase (lacZ) (5,36), green fluorescent protein (GFP) (18,26,29), and luciferase operon (lux) (1,13,19,26,27,35,41) reporter genes. For the comparative studies using reporter constructs, especially under conditions of global repression of gene expression, it is highly beneficial to use an additional reporter gene as a reference.…”

mentioning

confidence: 99%

Attenuation-Based Dual-Fluorescent-Protein Reporter for Screening Translation Inhibitors

Osterman

Prokhorova

Sysoev

et al. 2012

Antimicrob Agents Chemother

View full text Add to dashboard Cite

A reporter construct was created on the basis of the transcription attenuator region of the Escherichia coli tryptophan operon. Dual-fluorescent-protein genes for red fluorescent protein and cerulean fluorescent protein were used as a sensor and internal control of gene expression. The sequence of the attenuator was modified to avoid tryptophan sensitivity while preserving sensitivity to ribosome stalling. Antimicrobial compounds which cause translation arrest at the stage of elongation induce the reporter both in liquid culture and on an agar plate. This reporter could be used for high-throughput screening of translation inhibitors.

show abstract

“…In these "reverse" approaches, luciferase expression provides a control signal which is inhibited when the cells are incubated with transcriptional/translational inhibitors [37][38][39]. In these "reverse" approaches, luciferase expression provides a control signal which is inhibited when the cells are incubated with transcriptional/translational inhibitors [37][38][39].…”

Section: Approach Main Advantages Main Disadvantagesmentioning

confidence: 99%

Whole Cell Strategies Based on lux Genes for High Throughput Applications Toward New Antimicrobials

Karp

Galluzzi²

2006

CCHTS

Self Cite

View full text Add to dashboard Cite

The discovery/development of novel drug candidates has witnessed dramatic changes over the last two decades. Old methods to identify lead compounds are not suitable to screen wide libraries generated by combinatorial chemistry techniques. High throughput screening (HTS) has become irreplaceable and hundreds of different approaches have been described. Assays based on purified components are flanked by whole cell-based assays, in which reporter genes are used to monitor, directly or indirectly, the influence of a chemical over the metabolism of living cells. The most convenient and widely used reporters for real-time measurements are luciferases, light emitting enzymes from evolutionarily distant organisms. Autofluorescent proteins have been also extensively employed, but proved to be more suitable for end-point measurements, in situ applications - such as the localization of fusion proteins in specific subcellular compartments - or environmental studies on microbial populations. The trend toward miniaturization and the technical advances in detection and liquid handling systems will allow to reach an ultra high throughput screening (uHTS), with 100,000 of compounds routinely screened each day. Here we show how similar approaches may be applied also to the search for new and potent antimicrobial agents.

show abstract

Amplified Detection of Transcriptional and Translational Inhibitors in Bioluminescent Escherichia coli K-12

Cited by 11 publications

References 26 publications

Low-Oxygen-Recovery Assay for High-Throughput Screening of Compounds against Nonreplicating Mycobacterium tuberculosis

Low-Oxygen-Recovery Assay for High-Throughput Screening of Compounds against Nonreplicating Mycobacterium tuberculosis

Attenuation-Based Dual-Fluorescent-Protein Reporter for Screening Translation Inhibitors

Whole Cell Strategies Based on lux Genes for High Throughput Applications Toward New Antimicrobials

Contact Info

Product

Resources

About