scholarly journals Dominant Role of Paraoxonases in Inactivation of the Pseudomonas aeruginosa Quorum-Sensing Signal N-(3-Oxododecanoyl)-l-Homoserine Lactone

2008 ◽  
Vol 76 (6) ◽  
pp. 2512-2519 ◽  
Author(s):  
John F. Teiber ◽  
Sven Horke ◽  
Donovan C. Haines ◽  
Puneet K. Chowdhary ◽  
Junhui Xiao ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Specific Activity ◽  
Dominant Role ◽  
High Specific Activity ◽  
Homoserine Lactone ◽  
Mouse Lung ◽  
Calcium Dependent ◽  
Protein Levels ◽  
Human Sera

ABSTRACT The pathogenic bacterium Pseudomonas aeruginosa causes serious infections in immunocompromised patients. N-(3-Oxododecanoyl)-l-homoserine lactone (3OC12-HSL) is a key component of P. aeruginosa's quorum-sensing system and regulates the expression of many virulence factors. 3OC12-HSL was previously shown to be hydrolytically inactivated by the paraoxonase (PON) family of calcium-dependent esterases, consisting of PON1, PON2, and PON3. Here we determined the specific activities of purified human PONs for 3OC12-HSL hydrolysis, including the common PON1 polymorphic forms, and found they were in the following order: PON2 ≫ PON1192R > PON1192Q > PON3. PON2 exhibited a high specific activity of 7.6 ± 0.4 μmols/min/mg at 10 μM 3OC12-HSL, making it the best PON2 substrate identified to date. By use of class-specific inhibitors, approximately 85 and 95% of the 3OC12-HSL lactonase activity were attributable to PON1 in mouse and human sera, respectively. In mouse liver homogenates, the activity was metal dependent, with magnesium- and manganese-dependent lactonase activities comprising 10 to 15% of the calcium-dependent activity. In mouse lung homogenates, all of the activity was calcium dependent. The calcium-dependent activities were irreversibly inhibited by extended EDTA treatment, implicating PONs as the major enzymes inactivating 3OC12-HSL. In human HepG2 and EA.hy 926 cell lysates, the 3OC12-HSL lactonase activity closely paralleled the PON2 protein levels after PON2 knockdown by small interfering RNA treatment of the cells. These findings suggest that PONs, particularly PON2, could be an important mechanism by which 3OC12-HSL is inactivated in mammals.

scholarly journals Azithromycin Inhibits MUC5AC Production Induced by the Pseudomonas aeruginosa Autoinducer N-(3-Oxododecanoyl) Homoserine Lactone in NCI-H292 Cells

2004 ◽  
Vol 48 (9) ◽  
pp. 3457-3461 ◽  
Author(s):  
Yoshifumi Imamura ◽  
Katsunori Yanagihara ◽  
Yohei Mizuta ◽  
Masafumi Seki ◽  
Hideaki Ohno ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Bacterial Infection ◽  
Core Protein ◽  
Homoserine Lactone ◽  
Signal Molecule ◽  
Diffuse Panbronchiolitis ◽  
Mucin Production ◽  
Protein Levels ◽  
Extracellular Signal

ABSTRACT The features of chronic airway diseases, including chronic bronchitis, cystic fibrosis, bronchiectasis, and diffuse panbronchiolitis, include chronic bacterial infection and airway obstruction by mucus. Pseudomonas aeruginosa is one of the most common pathogens in chronic lung infection, and quorum-sensing systems contribute to the pathogenesis of this disease. The quorum-sensing signal molecule [N-(3-oxododecanoyl) homoserine lactone (3O-C12-HSL)] not only regulates bacterial virulence but also is associated with the immune response. In this study, we investigated whether 3O-C12-HSL could stimulate the production of a major mucin core protein, MUC5AC. The effect of a macrolide on MUC5AC production was also studied. 3O-C12-HSL induced NCI-H292 cells to express MUC5AC at both the mRNA and the protein levels in time- and dose-dependent manners. A 15-membered macrolide, azithromycin, inhibited MUC5AC production that was activated by 3O-C12-HSL. 3O-C12-HSL induced extracellular signal-regulated kinase (ERK) 1/2 and I-κB phosphorylation in cells, and this induction was suppressed by azithromycin. 3O-C12-HSL-induced MUC5AC production was blocked by the ERK pathway inhibitor PD98059. Our findings suggest that the P. aeruginosa autoinducer 3O-C12-HSL contributes to excessive mucin production in chronic bacterial infection. Azithromycin seems to reduce this mucin production by interfering with intracellular signal transduction.

scholarly journals Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 712-723 ◽  
Author(s):  
Valérie Dekimpe ◽  
Eric Déziel
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Transcriptional Regulator ◽  
Homoserine Lactone ◽  
The Other ◽  
Virulence Determinants ◽  
Late Stationary Phase ◽  
Regulatory Systems ◽  
Specific Factors

Pseudomonas aeruginosa uses the two major quorum-sensing (QS) regulatory systems las and rhl to modulate the expression of many of its virulence factors. The las system is considered to stand at the top of the QS hierarchy. However, some virulence factors such as pyocyanin have been reported to still be produced in lasR mutants under certain conditions. Interestingly, such mutants arise spontaneously under various conditions, including in the airways of cystic fibrosis patients. Using transcriptional lacZ reporters, LC/MS quantification and phenotypic assays, we have investigated the regulation of QS-controlled factors by the las system. Our results show that activity of the rhl system is only delayed in a lasR mutant, thus allowing the expression of multiple virulence determinants such as pyocyanin, rhamnolipids and C4-homoserine lactone (HSL) during the late stationary phase. Moreover, at this stage, RhlR is able to overcome the absence of the las system by activating specific LasR-controlled functions, including production of 3-oxo-C12-HSL and Pseudomonas quinolone signal (PQS). P. aeruginosa is thus able to circumvent the deficiency of one of its QS systems by allowing the other to take over. This work demonstrates that the QS hierarchy is more complex than the model simply presenting the las system above the rhl system.

scholarly journals Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa

2008 ◽  
Vol 52 (10) ◽  
pp. 3648-3663 ◽  
Author(s):  
Mette E. Skindersoe ◽  
Morten Alhede ◽  
Richard Phipps ◽  
Liang Yang ◽  
Peter O. Jensen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Virulence Factor ◽  
Dna Microarrays ◽  
Underlying Mechanism ◽  
Homoserine Lactone ◽  
Reverse Transcription Pcr ◽  
Animal Infection ◽  
Beneficial Action ◽  
Severe Infections

ABSTRACT During infection, Pseudomonas aeruginosa employs bacterial communication (quorum sensing [QS]) to coordinate the expression of tissue-damaging factors. QS-controlled gene expression plays a pivotal role in the virulence of P. aeruginosa, and QS-deficient mutants cause less severe infections in animal infection models. Treatment of cystic fibrosis (CF) patients chronically infected with P. aeruginosa with the macrolide antibiotic azithromycin (AZM) has been demonstrated to improve the clinical outcome. Several studies indicate that AZM may accomplish its beneficial action in CF patients by impeding QS, thereby reducing the pathogenicity of P. aeruginosa. This led us to investigate whether QS inhibition is a common feature of antibiotics. We present the results of a screening of 12 antibiotics for their QS-inhibitory activities using a previously described QS inhibitor selector 1 strain. Three of the antibiotics tested, AZM, ceftazidime (CFT), and ciprofloxacin (CPR), were very active in the assay and were further examined for their effects on QS-regulated virulence factor production in P. aeruginosa. The effects of the three antibiotics administered at subinhibitory concentrations were investigated by use of DNA microarrays. Consistent results from the virulence factor assays, reverse transcription-PCR, and the DNA microarrays support the finding that AZM, CFT, and CPR decrease the expression of a range of QS-regulated virulence factors. The data suggest that the underlying mechanism may be mediated by changes in membrane permeability, thereby influencing the flux of N-3-oxo-dodecanoyl-l-homoserine lactone.

scholarly journals Quorum-Sensing Genes in Pseudomonas aeruginosa Biofilms: Their Role and Expression Patterns

2001 ◽  
Vol 67 (4) ◽  
pp. 1865-1873 ◽  
Author(s):  
Teresa R. De Kievit ◽  
Richard Gillis ◽  
Steve Marx ◽  
Chris Brown ◽  
Barbara H. Iglewski
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Homoserine Lactone ◽  
Lower Percentage ◽  
Spatial Studies ◽  
Green Fluorescent ◽  
Biofilm Development

ABSTRACT Acylated homoserine lactone molecules are used by a number of gram-negative bacteria to regulate cell density-dependent gene expression by a mechanism known as quorum sensing (QS). InPseudomonas aeruginosa, QS or cell-to-cell signaling controls expression of a number of virulence factors, as well as biofilm differentiation. In this study, we investigated the role played by the las and rhl QS systems during the early stages of static biofilm formation when cells are adhering to a surface and forming microcolonies. These studies revealed a marked difference in biofilm formation between the PAO1 parent and the QS mutants when glucose, but not citrate, was used as the sole carbon source. To further elucidate the contribution of lasI andrhlI to biofilm maturation, we utilized fusions to unstable green fluorescent protein in concert with confocal microscopy to perform real-time temporal and spatial studies of these genes in a flowing environment. During the course of 8-day biofilm development,lasI expression was found to progressively decrease over time. Conversely, rhlI expression remained steady throughout biofilm development but occurred in a lower percentage of cells. Spatial analysis revealed that lasI andrhlI were maximally expressed in cells located at the substratum and that expression decreased with increasing biofilm height. Because QS was shown previously to be involved in biofilm differentiation, these findings have important implications for the design of biofilm prevention and eradication strategies.

scholarly journals Alginate Oligosaccharide-Induced Modification of the lasI-lasR and rhlI-rhlR Quorum-Sensing Systems in Pseudomonas aeruginosa

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Alison A. Jack ◽  
Saira Khan ◽  
Lydia C. Powell ◽  
Manon F. Pritchard ◽  
Konrad Beck ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Laser Scanning ◽  
Homoserine Lactone ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Mass Spectrometry Analysis ◽  
Confocal Laser ◽  
Steric Interaction ◽  
Content Type

ABSTRACT Pseudomonas aeruginosa plays a major role in many chronic infections. Its ability to readily form biofilms contributes to its success as an opportunistic pathogen and its resistance/tolerance to antimicrobial/antibiotic therapy. A low-molecular-weight alginate oligomer (OligoG CF-5/20) derived from marine algae has previously been shown to impair motility in P. aeruginosa biofilms and disrupt pseudomonal biofilm assembly. As these bacterial phenotypes are regulated by quorum sensing (QS), we hypothesized that OligoG CF-5/20 may induce alterations in QS signaling in P. aeruginosa . QS regulation was studied by using Chromobacterium violaceum CV026 biosensor assays that showed a significant reduction in acyl homoserine lactone (AHL) production following OligoG CF-5/20 treatment (≥2%; P < 0.05). This effect was confirmed by liquid chromatography-mass spectrometry analysis of C 4 -AHL and 3-oxo-C 12 -AHL production (≥2%; P < 0.05). Moreover, quantitative PCR showed that reduced expression of both the las and rhl systems was induced following 24 h of treatment with OligoG CF-5/20 (≥0.2%; P < 0.05). Circular dichroism spectroscopy indicated that these alterations were not due to steric interaction between the AHL and OligoG CF-5/20. Confocal laser scanning microscopy (CLSM) and COMSTAT image analysis demonstrated that OligoG CF-5/20-treated biofilms had a dose-dependent decrease in biomass that was associated with inhibition of extracellular DNA synthesis (≥0.5%; P < 0.05). These changes correlated with alterations in the extracellular production of the pseudomonal virulence factors pyocyanin, rhamnolipids, elastase, and total protease ( P < 0.05). The ability of OligoG CF-5/20 to modify QS signaling in P. aeruginosa PAO1 may influence critical downstream functions such as virulence factor production and biofilm formation.

scholarly journals GidA Posttranscriptionally Regulates rhl Quorum Sensing in Pseudomonas aeruginosa

2009 ◽  
Vol 191 (18) ◽  
pp. 5785-5792 ◽  
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.

scholarly journals A rhlI 5′ UTR-Derived sRNA Regulates RhlR-Dependent Quorum Sensing in Pseudomonas aeruginosa

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Maureen K. Thomason ◽  
Maya Voichek ◽  
Daniel Dar ◽  
Victoria Addis ◽  
David Fitzgerald ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Regulatory Elements ◽  
Content Type ◽  
Reading Frame ◽  
Bacterial Physiology ◽  
Small Regulatory Rna ◽  
Key Enzyme ◽  
Opportunistic Human Pathogen

ABSTRACT N-Acyl homoserine lactone (AHL) quorum sensing (QS) controls expression of over 200 genes in Pseudomonas aeruginosa. There are two AHL regulatory systems: the LasR-LasI circuit and the RhlR-RhlI system. We mapped transcription termination sites affected by AHL QS in P. aeruginosa, and in doing so we identified AHL-regulated small RNAs (sRNAs). Of interest, we noted that one particular sRNA was located within the rhlI locus. We found that rhlI, which encodes the enzyme that produces the AHL N-butanoyl-homoserine lactone (C4-HSL), is controlled by a 5′ untranslated region (UTR)-derived sRNA we name RhlS. We also identified an antisense RNA encoded opposite the beginning of the rhlI open reading frame, which we name asRhlS. RhlS accumulates as wild-type cells enter stationary phase and is required for the production of normal levels of C4-HSL through activation of rhlI translation. RhlS also directly posttranscriptionally regulates at least one other unlinked gene, fpvA. The asRhlS appears to be expressed at maximal levels during logarithmic growth, and we suggest RhlS may act antagonistically to the asRhlS to regulate rhlI translation. The rhlI-encoded sRNAs represent a novel aspect of RNA-mediated tuning of P. aeruginosa QS. IMPORTANCE The opportunistic human pathogen Pseudomonas aeruginosa possesses multiple quorum sensing systems that regulate and coordinate production of virulence factors and adaptation to different environments. Despite extensive research, the regulatory elements that play a role in this complex network are still not fully understood. By using several RNA sequencing techniques, we were able to identify a small regulatory RNA we named RhlS. RhlS increases translation of RhlI, a key enzyme in the quorum sensing pathway, and represses the fpvA mRNA encoding one of the siderophore pyoverdine receptors. Our results highlight a new regulatory layer of P. aeruginosa quorum sensing and contribute to the growing understanding of the role regulatory RNAs play in bacterial physiology.

scholarly journals The Pseudomonas aeruginosa Lectins PA-IL and PA-IIL Are Controlled by Quorum Sensing and by RpoS

2000 ◽  
Vol 182 (22) ◽  
pp. 6401-6411 ◽  
Author(s):  
Klaus Winzer ◽  
Colin Falconer ◽  
Nachman C. Garber ◽  
Stephen P. Diggle ◽  
Miguel Camara ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Transcriptional Start Site ◽  
Immunoblot Analysis ◽  
Homoserine Lactone ◽  
Start Codon ◽  
Virulence Determinants ◽  
Putative Promoter Region ◽  
Consensus Sequences ◽  
Translational Start

ABSTRACT In Pseudomonas aeruginosa, many exoproduct virulence determinants are regulated via a hierarchical quorum-sensing cascade involving the transcriptional regulators LasR and RhlR and their cognate activators,N-(3-oxododecanoyl)-l-homoserine lactone (3O-C12-HSL) and N-butanoyl-l-homoserine lactone (C4-HSL). In this paper, we demonstrate that the cytotoxic lectins PA-IL and PA-IIL are regulated via quorum sensing. Using immunoblot analysis, the production of both lectins was found to be directly dependent on the rhl locus while, in alasR mutant, the onset of lectin synthesis was delayed but not abolished. The PA-IL structural gene, lecA, was cloned and sequenced. Transcript analysis indicated a monocistronic organization with a transcriptional start site 70 bp upstream of thelecA translational start codon. A lux box-type element together with RpoS (ςS) consensus sequences was identified upstream of the putative promoter region. InEscherichia coli, expression of alecA::lux reporter fusion was activated by RhlR/C4-HSL, but not by LasR/3O-C12-HSL, confirming direct regulation by RhlR/C4-HSL. Similarly, in P. aeruginosaPAO1, the expression of a chromosomallecA::lux fusion was enhanced but not advanced by the addition of exogenous C4-HSL but not 3O-C12-HSL. Furthermore, mutation of rpoS abolished lectin synthesis inP. aeruginosa, demonstrating that both RpoS and RhlR/C4-HSL are required. Although the C4-HSL-dependent expression of the lecA::lux reporter in E. coli could be inhibited by the presence of 3O-C12-HSL, this did not occur in P. aeruginosa. This suggests that, in the homologous genetic background, 3O-C12-HSL does not function as a posttranslational regulator of the RhlR/C4-HSL-dependent activation oflecA expression.

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

2003 ◽  
Vol 185 (7) ◽  
pp. 2080-2095 ◽  
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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