Effect of efflux pump inhibition on Pseudomonas aeruginosa transcriptome and virulence.

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-ß-naphthylamide (PAßN) 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 PAßN, depending on the strain. Transcriptomic and phenotypic analyses showed that the protection exerted by PAßN 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 PAßN impacts P. aeruginosa virulence, anti-virulence properties of EPIs are worthy to be explored, taking into account possible strain-specificity of their activity.

Functional characterization of the quorum sensing regulator RsaL in the plant-beneficial strain Pseudomonas putida WCS358.

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 σ(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.