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.

Efflux pumps of the resistance-nodulation-cell-division (RND) family increase antibiotic resistance in many bacterial pathogens, representing candidate targets for the development of antibiotic adjuvants. RND pumps have also been proposed to contribute to bacterial infection, implying that efflux pump inhibitors (EPIs) could also act as anti-virulence drugs. Nevertheless, EPIs are usually investigated only for their properties as antibiotic adjuvants, while their potential anti-virulence activity is seldom taken into account. In this study it is shown that RND efflux pumps contribute to Pseudomonas aeruginosa PAO1 pathogenicity in an insect model of infection, and that the well-characterized EPI Phe-Arg-β-naphthylamide (PAβN) is able to reduce in vivo virulence of the P. aeruginosa PAO1 laboratory strain, as well as of clinical isolates. The production of quorum sensing (QS) molecules and of QS-dependent virulence phenotypes is differentially affected by PAβN, depending on the strain. Transcriptomic and phenotypic analyses showed that the protection exerted by PAβN from P. aeruginosa PAO1 infection in vivo correlates with the down-regulation of key virulence genes (e.g. genes involved in iron and phosphate starvation). Since PAβN impacts P. aeruginosa virulence, anti-virulence properties of EPIs are worthy to be explored, taking into account possible strain-specificity of their activity.

In many bacteria, quorum sensing (QS) systems rely on a signal receptor and a synthase producing N-acyl-homoserine lactone(s) as the signal molecule(s). In some species, the rsaL gene, located between the signal receptor and synthase genes, encodes a repressor limiting signal synthase expression and hence signal molecule production. Here we investigate the molecular mechanism of action of the RsaL protein in the plant growth-promoting rhizobacterium Pseudomonas putida WCS358 (RsaL WCS ). In P. putida WCS358, RsaL WCS displayed a strong repressive effect on the promoter of the QS signal synthase gene, ppuI, while it did not repress the same promoter in Pseudomonas aeruginosa. DNase I protection assays showed that purified RsaL WCS specifically binds to ppuI on a DNA region overlapping the predicted 70 -binding site, but such protection was observed only at high protein concentrations. Accordingly, electrophoretic mobility shift assays showed that the RsaL WCS protein was not able to form stable complexes efficiently with a probe encompassing the ppuI promoter, while it formed stable complexes with the promoter of lasI, the gene orthologous to ppuI in P. aeruginosa. This difference seems to be dictated by the lower dyad symmetry of the RsaL WCS -binding sequence on the ppuI promoter relative to that on the lasI promoter. Comparison of the results obtained in vivo and in vitro suggests that RsaL WCS needs a molecular interactor/cofactor specific for P. putida WCS358 to repress ppuI transcription. We also demonstrate that RsaL WCS regulates siderophore-mediated growth limitation of plant pathogens and biofilm formation, two processes relevant for plant growth-promoting activity.T he cell-cell communication system termed quorum sensing (QS) allows bacterial populations to coordinate gene expression in response to cell density. It is believed that QS constitutes a central element for the social life of bacteria, conferring to the members of a bacterial community the ability to behave as an organized multicellular organism (2, 4).QS systems are based on the production and secretion of signal molecules that accumulate in the extracellular milieu. At a certain concentration, corresponding to the "quorum" cell density, the signal molecules are perceived by dedicated receptors that, once activated, trigger a physiological response concerted in the whole population. The majority of QS systems thus far described in Gram-negative proteobacteria rely on N-acyl homoserine lactones (N-acyl-HSL) as signal molecules; these QS systems have been found in more than 100 bacterial species (14).Several members of the Pseudomonas genus regulate their social behavior through N-acyl-HSL-based QS systems. In particular, N-acyl-HSL-dependent regulation plays a major role in the pathogenesis of the opportunistic pathogen Pseudomonas aeruginosa and is involved in plant colonization by several Pseudomonas rhizosphere strains (5,12,18,23,24).Pseudomonas putida WCS358 is a plant growth-promoting rhizobacterium (PGPR) originally isolated from the ...