The Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)Homoserine Lactone Contributes to Virulence and Induces Inflammation In Vivo

Pseudomonas aeruginosa has two well-characterized quorum-sensing systems, Las and Rhl. These systems are composed of LuxR-type proteins, LasR and RhlR, and two acyl homoserine lactone (AHL) synthases, LasI and RhlI. LasI catalyzes the synthesis of N-(3-oxododecanoyl)homoserine lactone (3O-C12-HSL), whereas RhlI catalyzes the synthesis of N-butyryl-homoserine lactone. There is little known about the importance of AHLs in vivo and what effects these molecules have on eukaryotic cells. In order to understand the role of AHLs in vivo, we first tested the effects that deletions of the synthase genes in P. aeruginosa had on colonization of the lung. We demonstrate that in an adult mouse acute-pneumonia model, deletion of the lasI gene or both the lasI and rhlI genes greatly diminished the ability of P. aeruginosa to colonize the lung. To determine whether AHLs have a direct effect on the host, we examined the effects of 3O-C12-HSL injected into the skin of mice. In this model, 3O-C(12)-HSL stimulated a significant induction of mRNAs for the cytokines interleukin-1alpha (IL-1alpha) and IL-6 and the chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemotactic protein 1, MIP-1beta, inducible protein 10, and T-cell activation gene 3. Additionally, dermal injections of 3O-C12-HSL also induced cyclooxygenase 2 (Cox-2) expression. The Cox-2 enzyme is important for the conversion of arachidonic acid to prostaglandins and is associated with edema, inflammatory infiltrate, fever, and pain. We also demonstrate that 3O-C12-HSL activates T cells to produce the inflammatory cytokine gamma interferon and therefore potentially promotes a Th1 environment. Induction of these inflammatory mediators in vivo is potentially responsible for the significant influx of white blood cells and subsequent tissue destruction associated with 3O-C12-HSL dermal injections. Therefore, the quorum-sensing systems of P. aeruginosa contribute to its pathogenesis both by regulating expression of virulence factors (exoenzymes and toxins) and by inducing inflammation.     

Mutation of pfm affects the adherence of Pseudomonas aeruginosa to host cells and the quorum sensing system

The Pseudomonas aeruginosa quorum sensing (QS) system is controlled by the signal molecules acyl homoserine lactones (AHLs) that are synthesized from acyl enoyl-acyl carrier proteins (acyl-ACPs) provided by the fatty acid biosynthesis cycle. Pfm (PA2950), an enoyl-CoA reductase, has previously been shown to affect swimming mobility and fatty acid biosynthesis. In this report, we further show that pfm influences bacterial adherence to human cells. Microarray assay results suggest that pfm affects bacterial adherence through its influence on the QS system. Further experiments confirmed that the pfm mutant strain produces significantly less QS signal molecules than the corresponding wild-type strain. Using strains Escherichia coli DH5α(pECP64, lasB'-lacZ) and E.?coli DH5α(pECP61.5, rhlA'-lacZ), biosensors for N-(3-oxododecanoyl) homoserine lactone (3O-C(12) -HSL) and N-butyryl homoserine lactone (C(4) -HSL), respectively, we found that pfm mutant strain produces decreased amounts of both signal molecules. Elastase activity and pyocyanin measurements further confirmed the reduced levels of 3O-C(12) -HSL and C(4) -HSL in the pfm mutant. Finally, bacterial virulence, as assessed by the Caenorhabditis elegans worm killing assay, is decreased in the pfm mutant. Taken together, these data indicate that pfm can be an important target for the control of P.?aeruginosa infectivity.     

Modeling the effect of acylated homoserine lactone antagonists in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a gram-negative bacterium that causes serious illnesses, particularly in immunocompromised individuals, often with a fatal outcome. The finding that the acylated homoserine lactone quorum sensing (QS) system controls the production of virulence factors in P. aeruginosa makes this system a possible target for antimicrobial therapy. It has been suggested that an N-(3-oxododecanoyl)-homoserine lactone (3O-C12-HSL) antagonist, a QS blocker (QSB), would interfere efficiently with the quorum sensing system in P. aeruginosa and thus reduce the virulence of this pathogen. In this work, a mathematical model of the QS system in P. aeruginosa has been developed. The model was used to virtually add 3O-C12-HSL antagonists that differed in their affinity for the receptor protein and for their ability to mediate degradation of the receptor. The model suggests that very small differences in these parameters for different 3O-C12-HSL antagonists can greatly affect the success of QSB based inhibition of the QS system in P. aeruginosa. Most importantly, it is proposed that the ability of the 3O-C12-HSL antagonist to mediate degradation of LasR is the core parameter for successful QSB based inhibition of the QS system in P. aeruginosa. Finally, this study demonstrates that QSBs can shift the system to a low steady state, corresponding to an uninduced state and thus, suggests that the use of 3O-C12-HSL antagonists may constitute a promising therapeutic approach against P. aeruginosa involved infections.     

Production of acylated homoserine lactones by different serotypes of Vibrio anguillarum both in culture and during infection of rainbow trout

Onehundred and forty-eight out of onehundred and fifty strains of Vibrio anguillarum isolated from vibriosis in Danish marine aquaculture produced bacterial communication signals, acylated homoserine lactones, eliciting a response in the Agrobacterium tumefaciens (pZLR4) monitoring system. One strain, a serotype O4, induced a strong response in the Chromobacterium violaceum (CV026) monitoring system. Profiles of AHLs determined by TLC separation revealed the presence of at least four AHLs and a compound similar to N-3-oxo-decanoyl homoserine lactone (3-oxo-C10-HSL) was present in all strains. The production rate of the presumed 3-oxo-C10-HSL followed the growth rate of V. anguillarum whereas the production rate of a small AHL (Rf value of 0.74) increased faster than the growth rate of V. anguillarum indicating autoinduction. AHLs were produced by all serotypes (O1 to O10) and by non-typable strains. During infection with V. anguillarum, AHLs could be extracted from liver, kidney and muscle of rainbow trout and AHLs were detected both in vitro and in vivo when cell numbers reached 10(7) per ml or gram. Preliminary investigations of interactions between AHLs and the fish immune system were carried out determining oxidative burst of fish macrophages exposed to 3-oxo-C10-HSL. No activation or suppression of the superoxide anion production in the head kidney macrophages was seen when treated with the AHL compound in concentrations of 1 nM-10 microM. Our data show that AHLs are produced by almost all V. anguillarum strains and that no clear pattern relating AHL production to disease or virulence appear.     

Identification of N-acylhomoserine lactones in mucopurulent respiratory secretions from cystic fibrosis patients

Pseudomonas aeruginosa and species of the Burkholderia cepacia complex are the primary bacterial pathogens contributing to lung disease in patients with cystic fibrosis. Quorum sensing systems using N-acyl homoserine lactone (AHL) signal molecules are involved in the regulation of a number of virulence factors in these species. Extracts of mucopurulent respiratory secretions from 13 cystic fibrosis patients infected with P. aeruginosa and/or strains of the B. cepacia complex were fractionated using reverse-phase fast pressure liquid chromatography and analyzed for the presence of AHLs using a traI-luxCDABE-based reporter that responds to AHLs with acyl chains ranging between 4 and 12 carbons. Using this assay system, a broad range of AHLs were detected and identified despite being present at low concentrations in limited sample volumes. N-(3-oxo-dodecanoyl)-l-homoserine lactone, N-(3-oxo-decanoyl)-l-homoserine lactone and N-octanoyl-l-homoserine lactone (OHL) were the AHLs most frequently identified. OHL and N-decanoyl-l-homoserine lactone were detected in nanomolar concentrations compared to picomolar amounts of the 3-oxo-derivatives of the AHLs identified.     

Pseudomonas aeruginosa quorum sensing molecule N-(3 oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells

Acyl-homoserine lactone (HSL) quorum sensing molecules play an important role in regulation of virulence gene expression in Pseudomonas aeruginosa. Here, we show that 3O-C(12)-HSL can disrupt barrier integrity in human epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER), increased paracellular flux, reduction in the expression and distribution of ZO-1 and occludin, and reorganization of F-actin. P. aeruginosa 3O-C(12)-HSL activate p38 and p42/44 kinases, and inhibition of these kinases partly prevented 3O-C(12)-HSL-induced changes in TER, paracellular flux and expression of occludin and ZO-1. These findings demonstrate that P. aeruginosa 3O-C(12)-HSL can modulate tight junction integrity of Caco-2 cells.     

Profiling acylated homoserine lactones in Yersinia ruckeri and influence of exogenous acyl homoserine lactones and known quorum-sensing inhibitors on protease production

AIMS: To profile the quorum-sensing (QS) signals in Yersinia ruckeri and to examine the possible regulatory link between QS signals and a typical QS-regulated virulence phenotype, a protease. METHODS AND RESULTS: Liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) showed that Y. ruckeri produced at least eight different acylated homoserine lactones (AHLs) with N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL) being the dominant molecule. Also, some uncommon AHL, N-(3-oxoheptanoyl)-L-homoserine lactone (3-oxo-C7-HSL) and N-(3-oxononanoyl)-L-homoserine lactone (3-oxo-C9-HSL), were produced. 3-oxo-C8-HSL was detected in organs from fish infected with Y. ruckeri. Protease production was significantly lower at temperatures above 23 degrees C than below although growth was faster at the higher temperatures. Neither addition of sterile filtered high-density Y. ruckeri culture supernatant nor the addition of pure exogenous AHLs induced protease production. Furthermore, three QS inhibitors (QSIs), sulfur-containing AHL analogues, did not inhibit protease production in Y. ruckeri. CONCLUSIONS: Exogenous AHL or sulfur-containing AHL analogues did not influence the protease production indicating that protease production may not be QS regulated in Y. ruckeri. SIGNIFICANCE AND IMPACT OF THE STUDY: The array of different AHLs produced indicates that the QS system of Y. ruckeri is complex and could involve several regulatory systems. In this case, neither AHLs nor QSI would be likely to directly affect a QS-regulated phenotype.     

The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone stimulates phagocytic activity in human macrophages through the p38 MAPK pathway

Quorum-sensing is an important mechanism for the regulation of bacteria-to-bacteria communication. Recent advances have demonstrated that the Pseudomonas aeruginosa signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone (3O-C(12)-HSL) is also a potent modulator of eukaryotic cells and may thus play an important role in the host response during P. aeruginosa infections. Little is known, however, about specific effects of 3O-C(12)-HSL molecules on human macrophages. To address this issue, we investigated the influence of 3O-C(12)-HSL on the phagocytic activity, production of reactive oxygen species, and activation of p38 and p42/44 MAPK signaling pathways in human macrophages. We show an effect of 3O-C(12)-HSL on the phagocytic capacity in human macrophages, which depends on concentration and time of exposure. When cells were exposed to 100 microM 3O-C(12)-HSL for 30 min or 1 h, the phagocytic activity increased 1.8 and 1.6 times, respectively. The 3O-C(12)-HSL treatments had no significant effect on the level of reactive oxygen species production. Furthermore, the p38 MAPK, but not the p42/44 MAPK, signaling pathway was activated in response to 3O-C(12)-HSL. In addition, specific blocking of p38 MAPK activation with 10 microM SB 203580 prevented the 3O-C(12)-HSL-induced increase in the phagocytic activity. These findings demonstrate that the bacterial quorum-sensing can play a significant role also in regulation of macrophage activity during infections caused by P. aeruginosa.     

Type 1 and type 2 responses to Leishmania major

Pseudomonas aeruginosa and Burkholderia cepacia cause destructive lung disease in cystic fibrosis (CF) patients. Both pathogens employ 'quorum sensing', i.e. cell-to-cell communication, via diffusible N-acyl-L-homoserine lactone (AHL) signal molecules, to regulate the production of a number of virulence determinants in vitro. However, to date, evidence that quorum sensing systems are functional and play a role in vivo is lacking. This study presents the first direct evidence for the presence of AHLs in CF sputum. A total of 42 samples from 25 CF patients were analysed using lux-based Escherichia coli AHL biosensors. AHLs were detected in sputum from patients colonised by P. aeruginosa or B. cepacia but not Staphylococcus aureus. Furthermore, using liquid chromatography-mass spectrometry and thin layer chromatography, we confirmed the presence of N-hexanoylhomoserine lactone and N-(3-oxododecanoyl)homoserine lactone respectively in sputum samples from patients colonised by P. aeruginosa.     

P. aeruginosa quorum-sensing systems and virulence

Quorum sensing is an important mechanism for the regulation of genes in many Gram-negative and Gram-positive bacteria. In the opportunistic pathogen Pseudomonas aeruginosa, the absence of one or more components of the quorum-sensing system results in a significant reduction in virulence. Recent advances in the past year have demonstrated that the quorum-sensing signal molecule 3O-C(12)-HSL is also a potent stimulator of multiple eukaryotic cells and thus may alter the host response during P. aeruginosa infections. Therefore, via the regulation of multiple factors and the production of 3O-C(12)-HSL, quorum-sensing systems have a significant effect on the virulence of the bacteria and also on how the host responds to P. aeruginosa infections.     

Quorum Sensing in <Emphasis Type="Italic">Aeromonas</Emphasis> Species Isolated from Patients in Malaysia

Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C₆. Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.     

The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone

Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).     

Characterization of a phosphotriesterase-like lactonase from Sulfolobus solfataricus and its immobilization for disruption of quorum sensing

SsoPox, a bifunctional enzyme with organophosphate hydrolase and N-acyl homoserine lactonase activities from the hyperthermophilic archaeon Sulfolobus solfataricus, was overexpressed and purified from recombinant Pseudomonas putida KT2440 with a yield of 9.4 mg of protein per liter of culture. The enzyme has a preference for N-acyl homoserine lactones (AHLs) with acyl chain lengths of at least 8 carbon atoms, mainly due to lower K(m) values for these substrates. The highest specificity constant obtained was for N-3-oxo-decanoyl homoserine lactone (k(cat)/K(m) = 5.5 × 10(3) M(-1)·s(-1)), but SsoPox can also degrade N-butyryl homoserine lactone (C(4)-HSL) and N-oxo-dodecanoyl homoserine lactone (oxo-C(12)-HSL), which are important for quorum sensing in our Pseudomonas aeruginosa model system. When P. aeruginosa PAO1 cultures were grown in the presence of SsoPox-immobilized membranes, the production of C(4)-HSL- and oxo-C(12)-HSL-regulated virulence factors, elastase, protease, and pyocyanin were significantly reduced. This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled by quorum-sensing signals.     

Quorum-sensing molecules: Sampling,identification and characterization of N-acyl-homoserine lactone in Vibrio sp

Quorum sensing (QS) is a mechanism by which gram-negative bacteria regulate their gene expression by making use of cell density. QS is triggered by a small molecule known as an autoinducer. Typically, gram-negative bacteria such as Vibrio produce signaling molecules called acyl homoserine lactones (AHLs). However, their levels are very low, making them difficult to detect. We used thin layer chromatography (TLC) to examine AHLs in different Vibrio species, such as Vibrio alginolyticus, Vibrio parahemolyticus, and Vibrio cholerae, against a standard- Chromobacterium violaceum. Further, AHLs were characterised by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC–MS). C4-HSL (N- butanoyl- L- homoserine lactone), C6-HSL (N- hexanoyl- L- homoserine lactone), 3-oxo-C8-HSL (N-(3-Oxooctanoyl)-DL-homoserine lactone), C8-HSL (N- octanoyl- L- homoserine lactone), C₁10-HSL (N- decanoyl- L- homoserine lactone), C12-HSL (N- dodecanoyl- L- homoserine lactone) and C14-HSL (N- tetradecanoyl- L- homoserine lactone) were identified from Vibrio. These results may provide a basis for blocking the AHL molecules of Vibrio, thereby reducing their pathogenicity and eliminating the need for antimicrobials.     

Profiling of acylated homoserine lactones of Vibrio anguillarum in vitro and in vivo: Influence of growth conditions and serotype

Vibrio anguillarum produces several interlinked acylated homoserine lactone (AHL) signal molecules which may influence expression of its virulence factors such as exoprotease production and biofilm formation. Using both thin layer chromatography and HPLC-high resolution mass spectrometry (HPLC-HRMS), we demonstrate in this study that the same types of AHLs are produced by many serotypes of V. anguillarum and that altering in vitro growth conditions (salinity, temperature and iron concentration) has little influence on the AHL-profile. Most strains produced N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) and N-(3-hydroxy-hexanoyl)-l-homoserine lactone (3-hydroxy-C6-HSL) as the dominant molecules. Also, two spots with AHL activity appeared on TLC plates, which could not be identified as AHL structures. Trace amounts of N-(3-hydroxy-octanoyl)-l-homoserine lactone, N-(3-hydroxy-decanoyl)-l-homoserine lactone and N-(3-hydroxy-dodecanoyl)-l-homoserine lactone (3-hydroxy-C8-HSL, 3-hydroxy-C10-HSL and 3-oxo-C12-HSL, respectively) were also detected by HPLC-HRMS analysis from in vitro cultures. Most studies of quorum sensing (QS) systems have been conducted in vitro, the purpose of our study was to determine if the same acylated homoserine lactones were produced in vivo during infection. Extracts from infected fish were purified using several solid phase extraction strategies to allow chromatographic detection and separation by both TLC and HLPC-HRMS. 3-oxo-C10-HSL and 3-hydroxy-C6-HSL were detected in organs from fish dying from vibriosis, however, compared to in vitro culturing where 3-oxo-C10-HSL is the dominant molecule, 3-hydroxy-C6-HSL was prominent in the infected fish tissues. Hence, the balance between the QS systems may be different during infection compared to in vitro cultures. For future studies of QS systems and the possible specific interference with expression of virulence factors, in vitro cultures should be optimised to reflect the in vivo situation.