Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa.

Antibiotics are becoming less effective in treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa. Antimicrobial therapies based on the inhibition of specific virulence-related traits, as opposed to growth inhibitors, constitute an innovative and appealing approach to tackle the threat of P. aeruginosa infections. The twin-arginine translocation (Tat) pathway plays an important role in the pathogenesis of P. aeruginosa, and constitutes a promising target for the development of anti-pseudomonal drugs. In this study we developed and optimized a whole-cell, one-well assay, based on native phospholipase C activity, to identify compounds active against the Tat system. Statistical robustness, sensitivity and consequently suitability for high-throughput screening (HTS) were confirmed by a dry run/pre-screening test scoring a Z' of 0.82 and a signal-to-noise ratio of 49. Using this assay, we evaluated ca. 40,000 molecules and identified 59 initial hits as possible Tat inhibitors. Since phospholipase C is exported into the periplasm by Tat, and subsequently translocated across the outer membrane by the type II secretion system (T2SS), our assay could also identify T2SS inhibitors. To validate our hits and discriminate between compounds that inhibited either Tat or T2SS, two separate counter assays were developed and optimized. Finally, three Tat inhibitors and one T2SS inhibitor were confirmed by means of dose-response analysis and additional counter and confirming assays. Although none of the identified inhibitors was suitable as a lead compound for drug development, this study validates our assay as a simple, efficient, and HTS compatible method for the identification of Tat and T2SS inhibitors.

Repurposing the antimycotic drug flucytosine for suppression of Pseudomonas aeruginosa pathogenicity.

Although antibiotic resistance represents a public health emergency, the pipeline of new antibiotics is running dry. Repurposing of old drugs for new clinical applications is an attractive strategy for drug development. We used the bacterial pathogen Pseudomonas aeruginosa as a target for the screening of antivirulence activity among marketed drugs. We found that the antimycotic agent flucytosine inhibits the expression of the iron-starvation σ-factor PvdS, thereby repressing the production of major P. aeruginosa virulence factors, namely pyoverdine, PrpL protease, and exotoxin A. Flucytosine administration at clinically meaningful dosing regimens suppressed P. aeruginosa pathogenicity in a mouse model of lung infection. The in vitro and in vivo activity of flucytosine against P. aeruginosa, combined with its desirable pharmacological properties, paves the way for clinical trials on the anti-P. aeruginosa efficacy of flucytosine in humans.

New life for an old drug: the anthelmintic drug niclosamide inhibits Pseudomonas aeruginosa quorum sensing.

The need for novel antibacterial strategies and the awareness of the importance of quorum sensing (QS) in bacterial infections have stimulated research aimed at identifying QS inhibitors (QSIs). However, clinical application of QSIs identified so far is still distant, likely due to their unsuitability for use in humans. A promising way to overcome this problem is searching for anti-QS side activity among the thousands of drugs approved for clinical use in the treatment of different diseases. Here, we applied this strategy to the search for QSIs, by screening a library of FDA-approved compounds for their ability to inhibit the QS response in the Gram-negative pathogen Pseudomonas aeruginosa. We found that the anthelmintic drug niclosamide strongly inhibits the P. aeruginosa QS response and production of acyl-homoserine lactone QS signal molecules. Microarray analysis showed that niclosamide affects the transcription of about 250 genes, with a high degree of target specificity toward the QS-dependent regulon. Phenotypic assays demonstrated that niclosamide suppresses surface motility and production of the secreted virulence factors elastase, pyocyanin, and rhamnolipids, and it reduces biofilm formation. In accordance with the strong antivirulence activity disclosed in vitro, niclosamide prevented P. aeruginosa pathogenicity in an insect model of acute infection. Besides the finding that an FDA-approved drug has a promising antivirulence activity against one of the most antibiotic-resistant bacterial pathogens, this work provides a proof of concept that a lateral anti-QS activity can be detected among drugs already used in humans, validating a new approach to identify QSIs that could easily move into clinical applications.

A multitask biosensor for micro-volumetric detection of N-3-oxo-dodecanoyl-homoserine lactone quorum sensing signal.

N-3-oxo-dodecanoyl-homoserine lactone (3OC(12)-HSL) is the main quorum sensing (QS) signal produced by the human pathogen Pseudomonas aeruginosa, a major cause of hard-to-treat nosocomial infections and years-lasting chronic biofilm infections in the lungs of cystic fibrosis (CF) patients. 3OC(12)-HSL-dependent QS is considered a promising target for novel anti-pseudomonads drugs. However, the screening systems employed to date for the identification of QS inhibitors (QSI) were aimed at the identification of inhibitors of 3OC(12)-HSL signaling rather than of the synthesis or the export of this molecule. Moreover, the low concentration of 3OC(12)-HSL in CF sputum has hampered large scale studies aimed at addressing the role of this molecule in the CF lung infection. Here we describe the construction and characterization of PA14-R3, a new whole-cell biosensor for the quantitative detection of 3OC(12)-HSL. PA14-R3 provides fast and direct quantification of 3OC(12)-HSL over a wide range of concentrations (from pM to µM), and proved to be an easy-to-handle, cost-effective and reliable biosensor for high-throughput screening of 3OC(12)-HSL levels in samples of different origin, including CF sputum. Moreover, the specific features of PA14-R3 made it possible to develop and validate a novel high-throughput screening system for QSI based on the co-cultivation of PA14-R3 with the PA14 wild-type strain. With respect to previous screening systems for QSI, this approach has the advantage of being cost-effective and allowing the identification of compounds targeting, besides 3OC(12)-HSL signaling, any cellular process critical for QS response, including 3OC(12)-HSL synthesis and secretion.