Elisabetta Zennaro

RsaL provides quorum sensing homeostasis and functions as a global regulator of gene expression in Pseudomonas aeruginosa

The quorum sensing (QS) signalling system of Pseudomonas aeruginosa controls many important functions, including virulence. Although the production of the QS signal molecule N‐3‐oxo‐dodecanoyl‐homoserine lactone (3OC12‐HSL) is positively autoregulated, its concentration reaches a steady level long before stationary phase. The RsaL protein represses transcription of the lasI signal synthase gene, and thus reduces QS signal production. We show that RsaL binds simultaneously with LasR to the rsaL‐lasI bidirectional promoter thereby preventing the LasR‐dependent activation of both genes. In an rsaL mutant, 3OC12‐HSL production continues to increase throughout growth. Thus RsaL provides homeostasis by functioning in opposition to LasR and limiting 3OC12‐HSL production to a physiological concentration. Furthermore, transcription profiling revealed that RsaL regulates 130 genes independent of its effect on QS signal molecule production, including genes involved in virulence. We show that RsaL can repress pyocyanin and hydrogen cyanide virulence genes in two ways: directly, by binding to their promoters, and indirectly, by decreasing levels of the signals for their QS signal‐dependent transcription. These investigations highlight the importance of RsaL as a global regulator of P. aeruginosa physiology that provides a counterbalance to 3OC12‐HSL‐dependent gene activation via multiple mechanisms.

The Quorum-Sensing Negative Regulator RsaL of Pseudomonas aeruginosa Binds to the lasI Promoter

A mutation in the rsaL gene of Pseudomonas aeruginosa produces dramatically higher amounts of N-acyl homoserine lactone with respect to the wild type, highlighting the key role of this negative regulator in controlling quorum sensing (QS) in this opportunistic pathogen. The DNA binding site of the RsaL protein on the rsaL-lasI bidirectional promoter partially overlaps the binding site of the LasR protein, consistent with the hypothesis that RsaL and LasR could be in binding competition on this promoter. This is the first direct demonstration that RsaL acts as a QS negative regulator by binding to the lasI promoter.

New Life for an Old Drug: the Anthelmintic Drug Niclosamide Inhibits Pseudomonas aeruginosa Quorum Sensing

ABSTRACT 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.

Physiological Analysis of the Expression of the Styrene Degradation Gene Cluster in Pseudomonas fluorescens ST

ABSTRACT The effects of different carbon sources on expression of the styrene catabolism genes in Pseudomonas fluorescens ST were analyzed by using a promoter probe vector, pPR9TT, which contains transcription terminators upstream and downstream of the β-galactosidase reporter system. Expression of the promoter of thestySR operon, which codes for the styrene two-component regulatory system, was found to be constitutive and not subject to catabolite repression. This was confirmed by the results of an analysis of the stySR transcript in P. fluorescensST cells grown on different carbon sources. The promoter of the operon of the upper pathway, designated PstyA, was induced by styrene and repressed to different extents by organic acids or carbohydrates. In particular, cells grown on succinate or lactate in the presence of styrene started to exhibit β-galactosidase activity during the mid-exponential growth phase, before the preferred carbon sources were depleted, indicating that there is a threshold succinate and lactate concentration which allows induction of styrene catabolic genes. In contrast, cells grown on glucose, acetate, or glutamate and styrene exhibited a diauxic growth curve, and β-galactosidase activity was detected only after the end of the exponential growth phase. In each experiment the reliability of the reporter system constructed was verified by comparing the β-galactosidase activity and the activity of the styrene monooxygenase encoded by the first gene of the styrene catabolic operon.

Contribution of the RsaL global regulator to Pseudomonas aeruginosa virulence and biofilm formation

In Pseudomonas aeruginosa, acyl-homoserine-lactone quorum sensing (acyl-HSL QS) regulates the expression of virulence factors and biofilm formation in response to cell density. The RsaL protein represses transcription of the lasI gene, encoding the 3OC(12)-HSL signal synthase. The level of 3OC(12)-HSL is 10-fold higher in an rsaL mutant than in the wild type. In this work, we studied the effect of 3OC(12)-HSL overproduction caused by the rsaL mutation by comparing the transcriptional profiles and virulence-related phenotypes of a P. aeruginosa rsaL mutant and its wild-type parent. Results showed that the rsaL mutant overproduces secreted virulence factors (pyocyanin, elastase, hemolysins), displays increased twitching and swarming motility and is hypervirulent compared with the wild type. Interestingly, the rsaL mutant is impaired in biofilm formation. Taken together, these results suggest that RsaL could be important in the transition of P. aeruginosa from a planktonic to a sessile life style and in chronic infections, characterized by biofilm formation and limited virulence factor production.

Styrene-catabolism regulation in Pseudomonas fluorescens ST: Phosphorylation of StyR induces dimerization and cooperative DNA-binding

Styrene is an important chemical extensively used in the petrochemical and polymer industries. In Pseudomonas fluorescens ST, styrene metabolism is controlled by a two-component regulatory system, very uncommon in the degradation of aromatic compounds. The two-component regulatory proteins StyS and StyR regulate the expression of the styABCD operon, which codes for styrene degradation. StyS corresponds to the sensor kinase and StyR to the response regulator, which is essential for the activation of PstyA, the promoter of the catabolic operon. In two-component systems, the response regulator is phosphorylated by the cognate sensor kinase. Phosphorylation activates the response regulator, inducing DNA-binding. The mechanism underlying this activation has been reported only for a very few response regulators. Here, the effect of phosphorylation on the oligomeric state and on the DNA-binding properties of StyR has been investigated. Phosphorylation induces dimerization of StyR, the affinity of dimeric StyR for the target DNA is higher than that of the monomer, moreover dimeric StyR binding to the DNA target is cooperative. Furthermore, StyR oligomerization may be driven by the DNA target. This is the first direct demonstration that StyR response regulator binds to the PstyA promoter.

The Pseudomonas Quorum-Sensing Regulator RsaL Belongs to the Tetrahelical Superclass of H-T-H Proteins

In the opportunistic human pathogen Pseudomonas aeruginosa, quorum sensing (QS) is crucial for virulence. The RsaL protein directly represses the transcription of lasI, the synthase gene of the main QS signal molecule. On the basis of sequence homology, RsaL cannot be predicted to belong to any class of characterized DNA-binding proteins. In this study, an in silico model of the RsaL structure was inferred showing that RsaL belongs to the tetrahelical superclass of helix-turn-helix proteins. The overall structure of RsaL is very similar to the N-terminal domain of the lambda cI repressor and to the POU-specific domain of the mammalian transcription factor Oct-1 (Oct-1 POUs). Moreover, residues of Oct-1 POUs important for structural stability and/or DNA binding are conserved in the same positions in RsaL and in its homologs found in GenBank. These residues were independently replaced with Ala, and the activities of the mutated variants of RsaL were compared to that of the wild-type counterpart in vivo by complementation assays and in vitro by electrophoretic mobility shift assays. The results validated the RsaL in silico model and showed that residues Arg 20, Gln 38, Ser 42, Arg 43, and Glu 45 are important for RsaL function. Our data indicate that RsaL could be the founding member of a new protein family within the tetrahelical superclass of helix-turn-helix proteins. Finally, the minimum DNA sequence required for RsaL binding on the lasI promoter was determined, and our data support the hypothesis that RsaL binds DNA as a dimer.

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.

Resistance to Glycopeptide Antibiotics in the Teicoplanin Producer Is Mediated by van Gene Homologue Expression Directing the Synthesis of a Modified Cell Wall Peptidoglycan

ABSTRACT Glycopeptide resistance has been studied in detail in enterococci and staphylococci. In these microorganisms, high-level resistance is achieved by replacing the C-terminal d-alanyl-d-alanine of the nascent peptidoglycan with d-alanyl-d-lactate or d-alanyl-d-serine, thus reducing the affinities of glycopeptides for cell wall targets. Reorganization of the cell wall is directed by the expression of the van gene clusters. The identification of van gene homologs in the genomes of several glycopeptide-producing actinomycetes suggests the involvement of a similar self-resistance mechanism to avoid suicide. This report describes a comprehensive study of self-resistance in Actinoplanes teichomyceticus ATCC 31121, the producer of the clinically relevant glycopeptide teicoplanin. A. teichomyceticus ATCC 31121 showed a MIC of teicoplanin of 25 μg/ml and a MIC of vancomycin of 90 μg/ml during vegetative growth. The vanH, vanA, and vanX genes of A. teichomyceticus were found to be organized in an operon whose transcription was constitutive. Analysis of the UDP-linked peptidoglycan precursors revealed the presence of UDP-glycomuramyl pentadepsipeptide terminating in d-alanyl-d-lactate. No trace of precursors ending in d-alanyl-d-alanine was detected. Thus, the van gene complex was transcribed and expressed in the genetic background of A. teichomyceticus and conferred resistance to vancomycin and teicoplanin through the modification of cell wall biosynthesis. During teicoplanin production (maximum productivity, 70 to 80 μg/ml), the MIC of teicoplanin remained in the range of 25 to 35 μg/ml. Teicoplanin-producing cells were found to be tolerant to high concentrations of exogenously added glycopeptides, which were not bactericidal even at 5,000 μg/ml.

Dual role of response regulator StyR in styrene catabolism regulation.

ABSTRACT In Pseudomonas fluorescens ST, the promoter of the styrene catabolic operon, PstyA, is induced by styrene and repressed by the addition of preferred carbon sources. PstyA is regulated by the StyS/StyR two-component system. The integration host factor (IHF) also plays a positive role in PstyA regulation. Three distinct StyR binding sites, which have different affinities for this response regulator, have been characterized on PstyA. The high-affinity StyR binding site (STY2) is necessary for promoter activity. The DNA region upstream of STY2 contains a lower-affinity StyR binding site, STY1, that partially overlaps the IHF binding site. Deletion of this region, designated URE (upstream regulatory element), has a dual effect on the PstyA promoter, decreasing the styrene-dependent activity and partially relieving the glucose repression. The lowest-affinity StyR binding site (STY3) is located downstream of the transcription start point. Deletion of the URE region and inactivation of the STY3 site completely abolished glucose-mediated repression of PstyA. In the proposed model StyR can act either as an activator or as a repressor, depending on which sites it occupies in the different growth conditions. We suggest that the cellular levels of phosphorylated StyR, as determined by StyS sensor kinase activity, and the interplay of this molecule with IHF modulate the activity of the promoter in different growth conditions.