Selection of the N-Acylhomoserine Lactone-Degrading Bacterium Alteromonas stellipolaris PQQ-42 and of Its Potential for Biocontrol in Aquaculture

The production of virulence factors by many pathogenic microorganisms depends on the intercellular communication system called quorum sensing, which involves the production and release of signal molecules known as autoinducers. Based on this, new-therapeutic strategies have emerged for the treatment of a variety of infections, such as the enzymatic degradation of signaling molecules, known as quorum quenching (QQ). In this study, we present the screening of QQ activity amongst 450 strains isolated from a bivalve hatchery in Granada (Spain), and the selection of the strain PQQ-42, which degrades a wide range of N-acylhomoserine lactones (AHLs). The selected strain, identified as Alteromonas stellipolaris, degraded the accumulation of AHLs and reduced the production of protease and chitinase and swimming motility of a Vibrio species in co-cultivation experiments in vitro. In the bio-control experiment, strain PQQ-42 significantly reduced the pathogenicity of Vibrio mediterranei VibC-Oc-097 upon the coral Oculina patagonica showing a lower degree of tissue damage (29.25 ± 14.63%) in its presence, compared to when the coral was infected with V. mediterranei VibC-Oc-097 alone (77.53 ± 13.22%). Our results suggest that this AHL-degrading bacterium may have biotechnological applications in aquaculture.

Characterization of N-acylhomoserine lactone-degrading bacteria associated with the Zingiber officinale (ginger) rhizosphere: Co-existence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia

Background Cell-to-cell communication (quorum sensing (QS)) co-ordinates bacterial behaviour at a population level. Consequently the behaviour of a natural multi-species community is likely to depend at least in part on co-existing QS and quorum quenching (QQ) activities. Here we sought to discover novelN-acylhomoserine lactone (AHL)-dependent QS and QQ strains by investigating a bacterial community associated with the rhizosphere of ginger (Zingiber officinale) growing in the Malaysian rainforest. Results By using a basal growth medium containing N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) as the sole source of carbon and nitrogen, the ginger rhizosphere associated bacteria were enriched for strains with AHL-degrading capabilities. Three isolates belonging to the generaAcinetobacter (GG2), Burkholderia (GG4) and Klebsiella (Se14) were identified and selected for further study. Strains GG2 and Se14 exhibited the broadest spectrum of AHL-degrading activities via lactonolysis while GG4 reduced 3-oxo-AHLs to the corresponding 3-hydroxy compounds. In GG2 and GG4, QQ was found to co-exist with AHL-dependent QS and GG2 was shown to inactivate both self-generated and exogenously supplied AHLs. GG2, GG4 and Se14 were each able to attenuate virulence factor production in both human and plant pathogens. Conclusions Collectively our data show that ginger rhizosphere bacteria which make and degrade a wide range of AHLs are likely to play a collective role in determining the QS-dependent phenotype of a polymicrobial community.

The global posttranscriptional regulator RsmA modulates production of virulence determinants and N-acylhomoserine lactones in Pseudomonas aeruginosa.

Posttranscriptional control is known to contribute to the regulation of secondary metabolism and virulence determinants in certain gram-negative bacteria. Here we report the isolation of a Pseudomonas aeruginosa gene which encodes a global translational regulatory protein, RsmA (regulator of secondary metabolites). Overexpression of rsmA resulted in a substantial reduction in the levels of extracellular products, including protease, elastase, and staphylolytic (LasA protease) activity as well as the PA-IL lectin, hydrogen cyanide (HCN), and the phenazine pigment pyocyanin. While inactivation of rsmA in P. aeruginosa had only minor effects on the extracellular enzymes and the PA-IL lectin, the production of HCN and pyocyanin was enhanced during the exponential phase. The influence of RsmA on N-acylhomoserine lactone-mediated quorum sensing was determined by assaying the levels of N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL) and N-butanoylhomoserine lactone (C4-HSL) produced by the rsmA mutant and the rsmA-overexpressing strain. RsmA exerted a negative effect on the synthesis of both 3-oxo-C12-HSL and C4-HSL, which was confirmed by using lasI and rhlI translational fusions. These data also highlighted the temporal expression control of the lasI gene, which was induced much earlier and to a higher level during the exponential growth phase in an rsmA mutant. To investigate whether RsmA modulates HCN production solely via quorum-sensing control, hcn translational fusions were employed to monitor the regulation of the cyanide biosynthesis genes (hcnABC). RsmA was shown to exert an additional negative effect on cyanogenesis posttranscriptionally by acting on a region surrounding the hcnA ribosome-binding site. This suggests that, in P. aeruginosa, RsmA functions as a pleiotropic posttranscriptional regulator of secondary metabolites directly and also indirectly by modulating the quorum-sensing circuitry.

Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules. RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P. aeruginosa. An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P. aeruginosa. A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P. aeruginosa. Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation. Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.

Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1.

Virulence in the opportunistic human pathogen Pseudomonas aeruginosa is controlled by cell density via diffusible signalling molecules ('autoinducers') of the N-acylhomoserine lactone (AHL) type. Two Bacillus sp. isolates (A23 and A24) with AHL-degrading activity were identified among a large collection of rhizosphere bacteria. From isolate A24 a gene was cloned which was similar to the aiiA gene, encoding an AHL lactonase in another Bacillus strain. Expression of the aiiA homologue from isolate A24 in P. aeruginosa PAO1 reduced the amount of the quorum sensing signal N-oxododecanoyl-L-homoserine lactone and completely prevented the accumulation of the second AHL signal, N-butyryl-L-homoserine lactone. This strongly reduced AHL content correlated with a markedly decreased expression and production of several virulence factors and cytotoxic compounds such as elastase, rhamnolipids, hydrogen cyanide and pyocyanin, and strongly reduced swarming. However, no effect was observed on flagellar swimming or on twitching motility, and aiiA expression did not affect bacterial adhesion to a polyvinylchloride surface. In conclusion, introduction of an AHL degradation gene into P. aeruginosa could block cell-cell communication and exoproduct formation, but failed to interfere with surface colonization.

Quorum-sensing-negative (lasR) mutants of Pseudomonas aeruginosa avoid cell lysis and death.

In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37 degrees C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to > or = 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh(-) phenotype, and five of these mutants, which were functionally complemented by lasR(+), had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants.

The 4-aza-S-ribosyl-L-homocysteine derivatives and the related gamma-lactam and azahemiacetal analogs: Synthesis, inhibition and quorum sensing activity

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence, critical for establishing infection. There are two major pathways of QS systems. Type 1 is species specific or intra-species communication in which N-acylhomoserine lactones (Gram-negative bacteria) or oligopeptides (Gram-positive bacteria) are employed as signaling molecules (autoinducer one). Type 2 is inter-species communication in which S-4,5-dihydroxy-2,3-pentanedione (DPD) or its borate esters are used as signaling molecules. The DPD is biosynthesized by LuxS enzyme from S-ribosylhomocysteine (SRH). Recent increase in prevalence of bacterial strains resistant to antibiotics emphasizes the need for the development of new generation of antibacterial agents. Interruption of QS by small molecules is one of the viable options as it does not affect bacterial growth but only virulence, leading to less incidence of microbial resistance. Thus, in this work, inhibitors of both N-acylhomoserine lactone (AHL) mediated intra-species and LuxS enzyme, involved in inter-species QS are targeted. The γ-lactam and their reduced cyclic azahemiacetal analogs, bearing the additional alkylthiomethyl substituent, were designed and synthesized targeting AHL mediated QS systems in P. aeruginosa and Vibrio harveyi. The γ-lactams with nonylthio or dodecylthio chains acted as inhibitors of las signaling in P. aeruginosa with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent were found to strongly inhibit both las and rhl signaling in P. aeruginosa at higher concentrations. However, lactam and their azahemiacetal analogs were found to be inactive in V. harveyi QS systems. The 4-aza-S-ribosyl-L-homocysteine (4-aza-SRH) analogs and 2-deoxy-2-substituted-S-ribosyl-L-homocysteine analogs were designed and synthesized targeting Bacillus subtilis LuxS enzyme. The 4-aza-SRH analogs in which oxygen in ribose ring is replaced by nitrogen were further modified at anomeric position to produce pyrrolidine, lactam, nitrone, imine and hemiaminal analogs. Pyrrolidine and lactam analogs which lack anomeric hydroxyl, acted as competitive inhibitors of LuxS enzyme with KI value of 49 and 37 µM respectively. The 2,3-dideoxy lactam analogs were devoid of activity. Such findings attested the significance of hydroxyl groups for LuxS binding and activity. Hemiaminal analog of SRH was found to be a time-dependent inhibitor with IC50 value of 60 µM.

Synthesis and evaluation of sesquiterpene lactone inhibitors of phospholipase A(2) from Bothrops jararacussu

Several sesquiterpene lactone were synthesized and their inhibitive activities on phospholipase A(2) (PLA(2)) from Bothrops jararacussu venom were evaluated. Compounds Lac01 and Lac02 were efficient against PLA(2) edema-inducing, enzymatic and myotoxic activities and it reduces around 85% of myotoxicity and around 70% of edema-inducing activity. Lac05-Lac08 presented lower efficiency in inhibiting the biological activities studied and reduce the myotoxic and edema-inducing activities around only 15%. The enzymatic activity was significantly reduced. The values of inhibition constants (K(1)) for Lac01 and Lac02 were approximately 740 mu M, and for compounds Lac05-Lac08 the inhibition constants were approximately 7.622-9.240 mu M. The enzymatic kinetic studies show that the sesquiterpene lactones inhibit PLA(2) in a non-competitive manner. Some aspects of the structure-activity relationships (topologic, molecular and electronic parameters) were obtained using ab initio quantum calculations and analyzed by chemometric methods (HCA and PCA). The quantum chemistry calculations show that compounds with a higher capacity of inhibiting PLA(2) (Lac01-Lac04) present lower values of highest occupied molecular orbital (HOMO) energy and molecular volume (VOL) and bigger values of hydrophobicity (LogP). These results indicate some topologic aspects of the binding site of sesquiterpene lactone derivatives and PLA(2). (C) 2010 Elsevier Ltd. All rights reserved.

Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors

Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip((R)) microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response.

The Quorum-Sensing Molecules Farnesol/Homoserine Lactone and Dodecanol Operate via Distinct Modes of Action in Candida albicans.

Living as a commensal, Candida albicans must adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects on C. albicans, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C(12)-homoserine lactone, a quorum-sensing molecule secreted by Pseudomonas aeruginosa, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of the Candida adenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving the C. albicans hyphal repressor, Sfl1p. Deletion of SFL1 did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing in C. albicans is mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that the Burkholderia cenocepacia diffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C(12)-homoserine lactone, may be used by other quorum-sensing molecules.

The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N-butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase.

The global activator GacA, a highly conserved response regulator in Gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. The gacA gene of Pseudomonas aeruginosa PAO1 was isolated and its role in cell-density-dependent gene expression was characterized. Mutational inactivation of gacA resulted in delayed and reduced formation of the cell-density signal N-butyryl-L-homoserine lactone (BHL), of the cognate transcriptional activator RhIR (VsmR), and of the transcriptional activator LasR, which is known to positively regulate RhIR expression. Amplification of gacA on a multicopy plasmid caused precocious and enhanced production of BHL, RhIR and LasR. In parallel, the gacA gene dosage markedly influenced the BHL/RhIR-dependent formation of the cytotoxic compounds pyocyanin and cyanide and the exoenzyme lipase. However, the concentrations of another known cell-density signal of P. aeruginosa, N-oxododecanoyl-L-homoserine lactone, did not always match BHL concentrations. A model accounting for these observations places GacA function upstream of LasR and RhIR in the complex, cell-density-dependent signal-transduction pathway regulating several exoproducts and virulence factors of P. aeruginosa via BHL.

Étude de l’impact de la charge de travail sur les indicateurs biologiques d’exposition de l’acétone et du styrène par la modélisation toxicocinétique

L’effort physique a été reconnu comme l’un des déterminants majeurs qui affecte l’absorption pulmonaire et la cinétique des solvants industriels, composés volatils très répandus dans le milieu de travail. L’objectif global de ce projet était de caractériser la relation entre divers niveaux de charge de travail et les concentrations biologiques de l’acétone et du styrène ou de ses métabolites utilisés comme des indicateurs biologiques de l’exposition (IBEs) à ces solvants. Des modèles pharmacocinétiques à base physiologique ont été développés et validés afin de reproduire une exposition professionnelle à l’acétone et au styrène, individuellement et en combinaison, durant une semaine complète de travail (8h/jour, 5 jours). Les simulations ont été effectuées suivant une exposition aux valeurs limite d’exposition (500 ppm et 20 ppm, respectivement) et à des charges de travail de 12,5 W (repos), 25 W et 50 W. Les valeurs prédites par les modèles ont été comparées aux valeurs de référence des IBEs actuels. Le niveau d’acétone dans l’urine obtenu à la fin du dernier quart de travail était 3,5 fois supérieur à la valeur au repos (28 mg/L) pour un effort de 50 W, tandis que les niveaux de styrène dans le sang veineux et de ses métabolites dans l’urine ont augmenté d’un facteur d’environ 3,0 en comparaison avec les valeurs au repos, respectivement de 0,17 mg/L et 144 mg/g créatinine. Pour une co-exposition à des concentrations de 20 ppm de styrène et 200 ppm d’acétone et à une activité physique de 50 W, les simulations ont montré une augmentation de 10% du styrène sanguin et une faible diminution de ses métabolites dans l’urine. Les valeurs simulées par les modèles pour l’acétone ou le styrène montrent que des travailleurs dont la charge de travail équivaut à plus de 25 W sont susceptibles d’avoir des concentrations internes pouvant dépasser les valeurs de référence des IBEs respectifs de ces solvants et peuvent être à risque. Les résultats soulignent ainsi l’importance de tenir compte de la charge de travail dans la détermination d’une valeur de référence pour la surveillance biologique de l’acétone et du styrène.

Towards the total synthesis of colletofragarones: constructing the macrocyclic lactone by high pressure-mediated intramolecular Diels-Alder reaction

We report herein an intramolecular Diels-Alder approach towards the construction of the macrocyclic lactone ring and the perhydrobenzofuran system of the colletofragarones, novel metabolites produced by fungi of the genus Colletotrichum that are responsible for inhibition of germination of the conidia in these species. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

The soft nucleophilicity of organotellurolates driving the S(N)2-type lactone ring-opening reaction

Lithium and magnesium organotellurolates were reacted with lactones producing the corresponding tellurocarboxylic acids. Treatment of the reaction mixture with lithium aluminum hydride allowed the isolation of the corresponding hydroxytellurides in a one-pot operation. (C) 2009 Published by Elsevier Ltd