Genetic and transcriptomic characteristics of RhlR-dependent quorum sensing in cystic fibrosis isolates of Pseudomonas aeruginosa

In people with the genetic disease cystic fibrosis (CF), bacterial infections involving the opportunistic pathogen Pseudomonas aeruginosa are a significant cause of morbidity and mortality. P. aeruginosa uses a cell-cell signaling mechanism called quorum sensing (QS) to regulate many virulence functions. One type of QS consists of acyl-homoserine lactone (AHL) signals produced by LuxI-type signal synthases, which bind a cognate LuxR-type transcription factor. In laboratory strains and conditions, P. aeruginosa employs two AHL synthase/receptor pairs arranged in a hierarchy, with the LasI/R system controlling the RhlI/R system and many downstream virulence factors. However, P. aeruginosa isolates with inactivating mutations in lasR are frequently isolated from chronic CF infections. We and others have shown that these isolates frequently use RhlR as the primary QS regulator. RhlR is rarely mutated in CF and environmental settings. We were interested if there were reproducible genetic characteristics of these isolates and if there was a central group of genes regulated by RhlR in all isolates. We examined five isolates and found signatures of adaptation common to CF isolates. We did not identify a common genetic mechanism to explain the switch from Las- to Rhl-dominated QS. We describe a core RhlR regulon encompassing 20 genes encoding 7 products. These results suggest a key group of QS-regulated factors important for pathogenesis of chronic infection, and position RhlR as a target for anti-QS therapeutics. Our work underscores the need to sample a diversity of isolates to understanding QS beyond what has been described in laboratory strains.

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Instantaneous Within-Patient Diversity of Pseudomonas aeruginosa Quorum-Sensing Populations from Cystic Fibrosis Lung Infections

Infection and Immunity ◽

10.1128/iai.00755-09 ◽

2009 ◽

Vol 77 (12) ◽

pp. 5631-5639 ◽

Cited By ~ 84

Author(s):

Cara N. Wilder ◽

Gopal Allada ◽

Martin Schuster

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Opportunistic Pathogen ◽

Homoserine Lactone ◽

Loss Of Function ◽

Selective Forces ◽

Lung Infections

ABSTRACT In the opportunistic pathogen Pseudomonas aeruginosa, acyl-homoserine lactone (acyl-HSL) quorum sensing (QS) regulates biofilm formation and expression of many extracellular virulence factors. Curiously, QS-deficient variants, often carrying mutations in the central QS regulator LasR, are frequently isolated from infections, particularly from cystic fibrosis (CF) lung infections. Very little is known about the proportion and diversity of these QS variants in individual infections. Such information is desirable to better understand the selective forces that drive the evolution of QS phenotypes, including social cheating and innate (nonsocial) benefits. To obtain insight into the instantaneous within-patient diversity of QS, we assayed a panel of 135 concurrent P. aeruginosa isolates from eight different adult CF patients (9 to 20 isolates per patient) for various QS-controlled phenotypes. Most patients contained complex mixtures of QS-proficient and -deficient isolates. Among all patients, deficiency in individual phenotypes ranged from 0 to about 90%. Acyl-HSL, sequencing, and complementation analyses of variants with global loss-of-function phenotypes revealed dependency upon the central QS circuitry genes lasR, lasI, and rhlI. Deficient and proficient isolates were clonally related, implying evolution from a common ancestor in vivo. Our results show that the diversity of QS types is high within and among patients, suggesting diverse selection pressures in the CF lung. A single selective mechanism, be it of a social or nonsocial nature, is unlikely to account for such heterogeneity. The observed diversity also shows that conclusions about the properties of P. aeruginosa QS populations in individual CF infections cannot be drawn from the characterization of one or a few selected isolates.

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Solubility and Bioactivity of the Pseudomonas Quinolone Signal Are Increased by a Pseudomonas aeruginosa-Produced Surfactant

Infection and Immunity ◽

10.1128/iai.73.2.878-882.2005 ◽

2005 ◽

Vol 73 (2) ◽

pp. 878-882 ◽

Cited By ~ 60

Author(s):

M. Worth Calfee ◽

John G. Shelton ◽

James A. McCubrey ◽

Everett C. Pesci

Keyword(s):

Pseudomonas Aeruginosa ◽

Aqueous Solutions ◽

Opportunistic Pathogen ◽

Homoserine Lactone ◽

Cellular Functions ◽

Pseudomonas Quinolone Signal ◽

Serious Infections ◽

Enhanced Solubility ◽

Apoptosis Assay ◽

A Cell

ABSTRACT Pseudomonas aeruginosa is a gram-negative bacterium that causes serious infections in immunocompromised individuals and cystic fibrosis patients. This opportunistic pathogen controls many of its virulence factors and cellular functions through the activity of three cell-to-cell signals, N-(3-oxododecanoyl)-l-homoserine lactone, N-butyryl-l-homoserine lactone, and the Pseudomonas quinolone signal (PQS). The activity of these signals is dependent upon their ability to dissolve in and freely diffuse through the aqueous solution in which P. aeruginosa happens to reside. Despite this, our data indicated that PQS was relatively insoluble in aqueous solutions, which led us to postulate that P. aeruginosa could be producing a PQS-solubilizing factor. In this report, we show that the P. aeruginosa-produced biosurfactant rhamnolipid greatly enhances the solubility of PQS in aqueous solutions. The enhanced solubility of PQS led to an increase in PQS bioactivity, as measured by both a gene induction assay and an apoptosis assay. This is the first demonstration of the importance of a bacterial surfactant in the solubilization and bioactivity of a cell-to-cell signal.

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Disruption of Quorum Sensing and Virulence inBurkholderia cenocepaciaby a Structural Analogue of thecis-2-Dodecenoic Acid Signal

Applied and Environmental Microbiology ◽

10.1128/aem.00105-19 ◽

2019 ◽

Vol 85 (8) ◽

Cited By ~ 4

Author(s):

Chaoyu Cui ◽

Shihao Song ◽

Chunxi Yang ◽

Xiuyun Sun ◽

Yutong Huang ◽

...

Keyword(s):

Quorum Sensing ◽

Bacterial Infections ◽

Opportunistic Pathogen ◽

Homoserine Lactone ◽

Burkholderia Cenocepacia ◽

Enoic Acid ◽

Acyl Homoserine Lactone ◽

Structural Analogue ◽

Content Type ◽

Signal Production

ABSTRACTQuorum sensing (QS) signals are widely used by bacterial pathogens to control biological functions and virulence in response to changes in cell population densities.Burkholderia cenocepaciaemploys a molecular mechanism in which thecis-2-dodecenoic acid (namedBurkholderiadiffusiblesignalfactor [BDSF]) QS system regulatesN-acyl homoserine lactone (AHL) signal production and virulence by modulating intracellular levels of cyclic diguanosine monophosphate (c-di-GMP). Thus, inhibition of BDSF signaling may offer a non-antibiotic-based therapeutic strategy against BDSF-regulated bacterial infections. In this study, we report the synthesis of small-molecule mimics of the BDSF signal and evaluate their ability to inhibit BDSF QS signaling inB. cenocepacia. A novel structural analogue of BDSF, 14-Me-C16:Δ2(cis-14-methylpentadec-2-enoic acid), was observed to inhibit BDSF production and impair BDSF-regulated phenotypes inB. cenocepacia, including motility, biofilm formation, and virulence, while it did not inhibit the growth rate of this pathogen. 14-Me-C16:Δ2also reduced AHL signal production. Genetic and biochemical analyses showed that 14-Me-C16:Δ2inhibited the production of the BDSF and AHL signals by decreasing the expression of their synthase-encoding genes. Notably, 14-Me-C16:Δ2attenuated BDSF-regulated phenotypes in variousBurkholderiaspecies. These findings suggest that 14-Me-C16:Δ2could potentially be developed as a new therapeutic agent against pathogenicBurkholderiaspecies by interfering with their QS signaling.IMPORTANCEBurkholderia cenocepaciais an important opportunistic pathogen which can cause life-threatening infections in susceptible individuals, particularly in cystic fibrosis and immunocompromised patients. It usually employs two types of quorum sensing (QS) systems, including thecis-2-dodecenoic acid (BDSF) system andN-acyl homoserine lactone (AHL) system, to regulate virulence. In this study, we have designed and identified an unsaturated fatty acid compound (cis-14-methylpentadec-2-enoic acid [14-Me-C16:Δ2]) that is capable of interfering withB. cenocepaciaQS signaling and virulence. We demonstrate that 14-Me-C16:Δ2reduced BDSF and AHL signal production inB. cenocepacia. It also impaired QS-regulated phenotypes in variousBurkholderiaspecies. These results suggest that 14-Me-C16:Δ2could interfere with QS signaling in manyBurkholderiaspecies and might be developed as a new antibacterial agent.

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GidA Posttranscriptionally Regulates rhl Quorum Sensing in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00335-09 ◽

2009 ◽

Vol 191 (18) ◽

pp. 5785-5792 ◽

Cited By ~ 31

Author(s):

Rashmi Gupta ◽

Timothy R. Gobble ◽

Martin Schuster

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Flavin Adenine Dinucleotide ◽

Skim Milk ◽

Opportunistic Pathogen ◽

Homoserine Lactone ◽

Trna Modification ◽

Loss Of Function ◽

Novel Genes ◽

Lasa Protease

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa utilizes two interconnected acyl-homoserine lactone quorum-sensing (acyl-HSL QS) systems, LasRI and RhlRI, to regulate the expression of hundreds of genes. The QS circuitry itself is integrated into a complex network of regulation by other factors. However, our understanding of this network is still unlikely to be complete, as a comprehensive, saturating approach to identifying regulatory components has never been attempted. Here, we utilized a nonredundant P. aeruginosa PA14 transposon library to identify additional genes that regulate QS at the level of LasRI/RhlRI. We initially screened all 5,459 mutants for loss of function in one QS-controlled trait (skim milk proteolysis) and then rescreened attenuated candidates for defects in other QS phenotypes (LasA protease, rhamnolipid, and pyocyanin production) to exclude mutants defective in functions other than QS. We identified several known and novel genes, but only two novel genes, gidA and pcnB, affected all of the traits assayed. We characterized gidA, which exhibited the most striking QS phenotypes, further. This gene is predicted to encode a conserved flavin adenine dinucleotide-binding protein involved in tRNA modification. Inactivation of the gene primarily affected rhlR-dependent QS phenotypes such as LasA, pyocyanin, and rhamnolipid production. GidA affected RhlR protein but not transcript levels and also had no impact on LasR and acyl-HSL production. Overexpression of rhlR in a gidA mutant partially restored QS-dependent phenotypes. Taken together, these results indicate that GidA selectively controls QS gene expression posttranscriptionally via RhlR-dependent and -independent pathways.

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Microarray Analysis of Pseudomonas aeruginosa Quorum-Sensing Regulons: Effects of Growth Phase and Environment

Journal of Bacteriology ◽

10.1128/jb.185.7.2080-2095.2003 ◽

2003 ◽

Vol 185 (7) ◽

pp. 2080-2095 ◽

Cited By ~ 627

Author(s):

Victoria E. Wagner ◽

Daniel Bushnell ◽

Luciano Passador ◽

Andrew I. Brooks ◽

Barbara H. Iglewski

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Expression Patterns ◽

Antibiotic Sensitivity ◽

Opportunistic Pathogen ◽

Homoserine Lactone ◽

Medium Composition ◽

Logarithmic Phase ◽

Significant Differential Expression ◽

Biofilm Development

ABSTRACT Bacterial communication via quorum sensing (QS) has been reported to be important in the production of virulence factors, antibiotic sensitivity, and biofilm development. Two QS systems, known as the las and rhl systems, have been identified previously in the opportunistic pathogen Pseudomonas aeruginosa. High-density oligonucleotide microarrays for the P. aeruginosa PAO1 genome were used to investigate global gene expression patterns modulated by QS regulons. In the initial experiments we focused on identifying las and/or rhl QS-regulated genes using a QS signal generation-deficient mutant (PAO-JP2) that was cultured with and without added exogenous autoinducers [N-(3-oxododecanoyl) homoserine lactone and N-butyryl homoserine lactone]. Conservatively, 616 genes showed statistically significant differential expression (P ≤ 0.05) in response to the exogenous autoinducers and were classified as QS regulated. A total of 244 genes were identified as being QS regulated at the mid-logarithmic phase, and 450 genes were identified as being QS regulated at the early stationary phase. Most of the previously reported QS-promoted genes were confirmed, and a large number of additional QS-promoted genes were identified. Importantly, 222 genes were identified as being QS repressed. Environmental factors, such as medium composition and oxygen availability, eliminated detection of transcripts of many genes that were identified as being QS regulated.

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Function of the bacteriophytochrome BphP in the RpoS/Las quorum-sensing network of Pseudomonas aeruginosa

Microbiology ◽

10.1099/mic.0.049007-0 ◽

2011 ◽

Vol 157 (6) ◽

pp. 1651-1664 ◽

Cited By ~ 15

Author(s):

Katalin Barkovits ◽

Britta Schubert ◽

Sabrina Heine ◽

Maurice Scheer ◽

Nicole Frankenberg-Dinkel

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Response Regulator ◽

Red Light ◽

Regulatory System ◽

Global Gene Expression ◽

Opportunistic Pathogen ◽

Pleiotropic Effect ◽

Genes Encoding

The bacterial phytochrome of Pseudomonas aeruginosa (PaBphP) is an in vitro-active red/far-red light sensor histidine kinase of a two-component regulatory system. Despite solid biochemical data, its function in this heterotrophic, opportunistic pathogen is still unknown. Previous studies established that the genes encoding the two necessary phytochrome components BphO, a chromophore-producing haem oxygenase, and BphP, the apo-phytochrome, are co-transcribed in a bicistronic operon. Transcription has been shown to be induced in the stationary phase and to be dependent on the alternative sigma factor RpoS. Here we show an additional regulation of bphP expression through the quorum-sensing (QS) regulator LasR. This regulation is also reflected in a combination of expression profile experiments and proteome analyses of wild-type and phytochrome-deficient strains. While PaBphP has a pleiotropic effect on global gene expression, 66 % of the downregulated genes in the phytochrome mutant display a link to the Las QS system. Most of these genes seem to be indirectly regulated by LasR through BphP and the unknown response regulator BphR. A model of phytochrome function within the Las QS network is presented.

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Energy-Optimized Pharmacophore Coupled Virtual Screening in the Discovery of Quorum Sensing Inhibitors of LasR Protein of Pseudomonas aeruginosa

10.20944/preprints201910.0074.v1 ◽

2019 ◽

Author(s):

Zulkar Nain ◽

Sifat Bin Sayed ◽

Mohammad Minnatul Karim ◽

Md. Ariful Islam ◽

Utpal Kumar Adhikari

Keyword(s):

Pseudomonas Aeruginosa ◽

Virtual Screening ◽

Quorum Sensing ◽

Bacterial Infections ◽

Opportunistic Pathogen ◽

Vital Role ◽

Energy Calculation ◽

Quorum Sensing Inhibitors ◽

Binding Energy Calculation ◽

Standard Precision

Pseudomonas aeruginosa is an emerging opportunistic pathogen responsible for cystic fibrosis and nosocomial infections. In addition, empirical treatments are become inefficient due to their multiple-antibiotic resistance and extensive colonizing ability. Quorum sensing (QS) plays a vital role in the regulation of virulence factors in P. aeruginosa. Attenuation of virulence by QS inhibition could be an alternative and effective approach to control infections. Therefore, we sought to discover new QS inhibitors (QSIs) against LasR receptor in P. aeruginosa using chemoinformatics. Initially, a structure-based high-throughput virtual screening was performed using the LasR active site that identified 61404 relevant molecules. E-pharmacophore (ADAHH) screening of these molecules rendered 72 QSI candidates. In standard-precision docking, only 7 compounds were found as potential QSIs due to their higher binding affinity to LasR receptor (-7.53 to -10.32 kcal/mol compared to -7.43 kcal/mol of native ligands). The ADMET properties of these compounds were suitable to be QSIs. Later, extra-precision docking and binding energy calculation suggested ZINC19765885 and ZINC72387263 as the most promising QSIs. The dynamic simulation of the docked complexes showed good binding stability and molecular interactions. The current study suggested that these two compounds could be used in P. aeruginosa QS inhibition to combat bacterial infections.

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Evolution of the quorum sensing regulon in cooperating populations of Pseudomonas aeruginosa

10.1101/2021.07.01.450773 ◽

2021 ◽

Author(s):

Nicole E Smalley ◽

Amy L Schaefer ◽

Kyle L Asfahl ◽

Crystal Perez ◽

E Peter Greenberg ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Cell Communication ◽

Opportunistic Pathogen ◽

Quorum Quenching ◽

Homoserine Lactone ◽

Rna Seq ◽

Bacterial Strains ◽

Communication And Coordination

The bacterium Pseudomonas aeruginosa is an opportunistic pathogen and it thrives in many different saprophytic habitats. In this bacterium acyl-homoserine lactone quorum sensing (QS) can activate expression of over 100 genes, many of which code for extracellular products. P. aeruginosa has become a model for studies of cell-cell communication and coordination of cooperative activities. We hypothesized that long-term growth of bacteria under conditions where only limited QS-controlled functions were required would result in a reduction in the size of the QS-controlled regulon. To test this hypothesis, we grew P. aeruginosa for about 1000 generations in a condition in which expression of QS-activated genes is required for growth. We compared the QS regulons of populations after about 35 generations to those after about 1000 generations in two independent lineages by using quorum quenching and RNA-seq technology. In one evolved lineage the number of QS-activated genes identified was reduced by about 70% and in the other by about 45%. Our results lend important insights about the variations in the number of QS-activated genes reported for different bacterial strains and, more broadly, about the environmental histories of P. aeruginosa.

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The flavanone naringenin reduces the production of quorum sensing-controlled virulence factors in Pseudomonas aeruginosa PAO1

Microbiology ◽

10.1099/mic.0.049338-0 ◽

2011 ◽

Vol 157 (7) ◽

pp. 2120-2132 ◽

Cited By ~ 123

Author(s):

Olivier M. Vandeputte ◽

Martin Kiendrebeogo ◽

Tsiry Rasamiravaka ◽

Caroline Stévigny ◽

Pierre Duez ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Virulence Factors ◽

Pathogenic Bacteria ◽

Opportunistic Pathogen ◽

Homoserine Lactone ◽

Gene Products ◽

Strain Pao1 ◽

Preliminary Screening ◽

Mutant Strains

Preliminary screening of the Malagasy plant Combretum albiflorum for compounds attenuating the production of quorum sensing (QS)-controlled virulence factors in bacteria led to the identification of active fractions containing flavonoids. In the present study, several flavonoids belonging to the flavone, flavanone, flavonol and chalcone structural groups were screened for their capacity to reduce the production of QS-controlled factors in the opportunistic pathogen Pseudomonas aeruginosa (strain PAO1). Flavanones (i.e. naringenin, eriodictyol and taxifolin) significantly reduced the production of pyocyanin and elastase in P. aeruginosa without affecting bacterial growth. Consistently, naringenin and taxifolin reduced the expression of several QS-controlled genes (i.e. lasI, lasR, rhlI, rhlR, lasA, lasB, phzA1 and rhlA) in P. aeruginosa PAO1. Naringenin also dramatically reduced the production of the acylhomoserine lactones N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-l-homoserine lactone (C4-HSL), which is driven by the lasI and rhlI gene products, respectively. In addition, using mutant strains deficient for autoinduction (ΔlasI and ΔrhlI) and LasR- and RhlR-based biosensors, it was shown that QS inhibition by naringenin not only is the consequence of a reduced production of autoinduction compounds but also results from a defect in the proper functioning of the RlhR–C4-HSL complex. Widely distributed in the plant kingdom, flavonoids are known for their numerous and determinant roles in plant physiology, plant development and in the success of plant–rhizobia interactions, but, as shown here, some of them also have a role as inhibitors of the virulence of pathogenic bacteria by interfering with QS mechanisms.

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

Journal of Bacteriology ◽

10.1128/jb.188.2.815-819.2006 ◽

2006 ◽

Vol 188 (2) ◽

pp. 815-819 ◽

Cited By ~ 65

Author(s):

Giordano Rampioni ◽

Iris Bertani ◽

Elisabetta Zennaro ◽

Fabio Polticelli ◽

Vittorio Venturi ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Binding Site ◽

Opportunistic Pathogen ◽

Bidirectional Promoter ◽

Homoserine Lactone ◽

Negative Regulator ◽

Direct Demonstration ◽

Dna Binding Site

ABSTRACT 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.

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