LuxS-independent formation of AI-2 from ribulose-5-phosphate

<p>Abstract</p> <p>Background</p> <p>In many bacteria, the signal molecule AI-2 is generated from its precursor <it>S</it>-ribosyl-L-homocysteine in a reaction catalysed by the enzyme LuxS. However, generation of AI-2-like activity has also been reported for organisms lacking the <it>luxS </it>gene and the existence of alternative pathways for AI-2 formation in <it>Escherichia coli </it>has recently been predicted by stochastic modelling. Here, we investigate the possibility that spontaneous conversion of ribulose-5-phosphate could be responsible for AI-2 generation in the absence of <it>luxS</it>.</p> <p>Results</p> <p>Buffered solutions of ribulose-5-phosphate, but not ribose-5-phosphate, were found to contain high levels of AI-2 activity following incubation at concentrations similar to those reported <it>in vivo</it>. To test whether this process contributes to AI-2 formation by bacterial cells <it>in vivo</it>, an improved <it>Vibrio harveyi </it>bioassay was used. In agreement with previous studies, culture supernatants of <it>E. coli </it>and <it>Staphylococcus aureus luxS </it>mutants were found not to contain detectable levels of AI-2 activity. However, low activities were detected in an <it>E. coli pgi-eda-edd-luxS </it>mutant, a strain which degrades glucose entirely via the oxidative pentose phosphate pathway, with ribulose-5-phosphate as an obligatory intermediate.</p> <p>Conclusion</p> <p>Our results suggest that LuxS-independent formation of AI-2, via spontaneous conversion of ribulose-5-phosphate, may indeed occur <it>in vivo</it>. It does not contribute to AI-2 formation in wildtype <it>E. coli </it>and <it>S. aureus </it>under the conditions tested, but may be responsible for the AI-2-like activities reported for other organisms lacking the <it>luxS </it>gene.</p

Identification of FDA-Approved Drugs as Antivirulence Agents Targeting the pqs Quorum-Sensing System of Pseudomonas aeruginosa

Copyright © 2018 American Society for Microbiology. All Rights Reserved. The long-term use of antibiotics has led to the emergence of multidrug-resistant bacteria. A promising strategy to combat bacterial infections aims at hampering their adaptability to the host environment without affecting growth. In this context, the intercellular communication system quorum sensing (QS), which controls virulence factor production and biofilm formation in diverse human pathogens, is considered an ideal target. Here, we describe the identification of new inhibitors of the pqs QS system of the human pathogen Pseudomonas aeruginosa by screening a library of 1,600 U.S. Food and Drug Administration-approved drugs. Phenotypic characterization of ad hoc engineered strains and in silico molecular docking demonstrated that the antifungal drugs clotrimazole and miconazole, as well as an antibacterial compound active against Gram-positive pathogens, clofoctol, inhibit the pqs system, probably by targeting the transcriptional regulator PqsR. The most active inhibitor, clofoctol, specifically inhibited the expression of pqs-controlled virulence traits in P. aeruginosa, such as pyocyanin production, swarming motility, biofilm formation, and expression of genes involved in siderophore production. Moreover, clofoctol protected Galleria mellonella larvae from P. aeruginosa infection and inhibited the pqs QS system in P. aeruginosa isolates from cystic fibrosis patients. Notably, clofoctol is already approved for clinical treatment of pulmonary infections caused by Gram-positive bacterial pathogens; hence, this drug has considerable clinical potential as an antivirulence agent for the treatment of P. aeruginosa lung infections

Childhood fantasy play relates to adult socio‐emotional competence

Childhood fantasy play and creation of imaginary companions are thought to confer socio‐emotional benefits in children, but little is known about how they relate to socio‐emotional competence in adulthood. A total of 341 adults (81 males) aged 18 and above (M = 31.47, SD = 12.62) completed an online survey examining their fantasy play as a child, their childhood imaginary companion status, and their adult socio‐emotional competence. Adults who reported higher levels of childhood fantasy play were found to be significantly more prosocial, empathetic, and emotionally intelligent than their counterparts after controlling for demographic factors. Recall of a childhood imaginary companion, however, was significantly related only to higher scores for perspective‐taking and did not explain unique variance in any adult competence measure. Findings suggest that engagement in fantasy play during childhood may be a precursor to later socio‐emotional competence, while benefits previously associated with imaginary companions specifically may not extend into adulthood

Cross-kingdom signalling regulates spore germination in the moss <i>Physcomitrella patens</i>

Plants live in close association with microorganisms that can have beneficial or detrimental effects. The activity of bacteria in association with flowering plants has been extensively analysed. Bacteria use quorum-sensing as a way of monitoring their population density and interacting with their environment. A key group of quorum sensing molecules in Gram-negative bacteria are the N-acylhomoserine lactones (AHLs), which are known to affect the growth and development of both flowering plants, including crops, and marine algae. Thus, AHLs have potentially important roles in agriculture and aquaculture. Nothing is known about the effects of AHLs on the earliest-diverging land plants, thus the evolution of AHL-mediated bacterial-plant/algal interactions is unknown. In this paper, we show that AHLs can affect spore germination in a representative of the earliest plants on land, the Bryophyte moss Physcomitrella patens. Furthermore, we demonstrate that sporophytes of some wild isolates of Physcomitrella patens are associated with AHL-producing bacteria

Biotic inactivation of the Pseudomonas aeruginosa quinolone signal molecule

In Pseudomonas aeruginosa, quorum sensing (QS) regulates the production of secondary metabolites, many of which are antimicrobials that impact on polymicrobial community composition. Consequently, quenching QS modulates the environmental impact of P. aeruginosa. To identify bacteria capable of inactivating the QS signal molecule 2-heptyl-3- hydroxy-4(1H)-quinolone (PQS), a minimal medium containing PQS as the sole carbon source was used to enrich a Malaysian rainforest soil sample. This yielded an Achromobacter xylosoxidans strain (Q19) that inactivated PQS, yielding a new fluorescent compound (I-PQS) confirmed as PQS-derived using deuterated PQS. The I-PQS structure was elucidated using mass spectrometry and nuclear magnetic resonance spectroscopy as 2-heptyl-2-hydroxy-1,2-dihydroquinoline- 3,4-dione (HHQD). Achromobacter xylosoxidans Q19 oxidized PQS congeners with alkyl chains ranging from C1 to C5 and also N-methyl PQS, yielding the corresponding 2-hydroxy-1,2-dihydroquinoline-3,4- diones, but was unable to inactivate thePQSprecursor HHQ. This indicates that the hydroxyl group at position 3 in PQS is essential and that A. xylosoxidans inactivates PQS via a pathway involving the incorporation of oxygen at C2 of the heterocyclic ring. The conversion of PQS to HHQD also occurred on incubation with 12/17 A. xylosoxidans strains recovered from cystic fibrosis patients, with P. aeruginosa and with Arthrobacter, suggesting that formation of hydroxylated PQS may be a common mechanism of inactivation

Biotic inactivation of the Pseudomonas aeruginosa quinolone signal molecule

In Pseudomonas aeruginosa, quorum sensing (QS) regulates the production of secondary metabolites, many of which are antimicrobials that impact on polymicrobial community composition. Consequently, quenching QS modulates the environmental impact of P. aeruginosa. To identify bacteria capable of inactivating the QS signal molecule 2-heptyl-3- hydroxy-4(1H)-quinolone (PQS), a minimal medium containing PQS as the sole carbon source was used to enrich a Malaysian rainforest soil sample. This yielded an Achromobacter xylosoxidans strain (Q19) that inactivated PQS, yielding a new fluorescent compound (I-PQS) confirmed as PQS-derived using deuterated PQS. The I-PQS structure was elucidated using mass spectrometry and nuclear magnetic resonance spectroscopy as 2-heptyl-2-hydroxy-1,2-dihydroquinoline- 3,4-dione (HHQD). Achromobacter xylosoxidans Q19 oxidized PQS congeners with alkyl chains ranging from C1 to C5 and also N-methyl PQS, yielding the corresponding 2-hydroxy-1,2-dihydroquinoline-3,4- diones, but was unable to inactivate thePQSprecursor HHQ. This indicates that the hydroxyl group at position 3 in PQS is essential and that A. xylosoxidans inactivates PQS via a pathway involving the incorporation of oxygen at C2 of the heterocyclic ring. The conversion of PQS to HHQD also occurred on incubation with 12/17 A. xylosoxidans strains recovered from cystic fibrosis patients, with P. aeruginosa and with Arthrobacter, suggesting that formation of hydroxylated PQS may be a common mechanism of inactivation

The impaired quorum sensing response of MexAB?OprM efflux pump overexpressing mutants is not due to non?physiological efflux of 3?oxo?C12?HSL

Multidrug (MDR) efflux pumps are ancient and conserved molecular machineries with relevant roles in different aspects of the bacterial physiology, besides antibiotic resistance. In the case of the environmental opportunistic pathogen Pseudomonas aeruginosa, it has been shown that overexpression of different efflux pumps is linked to the impairment of the quorum sensing (QS) response. Nevertheless, the causes of such impairment are different for each analyzed efflux pump. Herein, we performed an in‐depth analysis of the QS‐mediated response of a P. aeruginosa antibiotic resistant mutant that overexpresses MexAB‐OprM. Although previous work claimed that this efflux pump extrudes the QS signal 3‐oxo‐C12‐HSL, we show otherwise. Our results evidence that the observed attenuation in the QS response when overexpressing this pump is related to an impaired production of alkyl quinolone QS signals, likely prompted by the reduced availability of one of their precursors, the octanoate. Together with previous studies, this indicates that, although the consequences of overexpressing efflux pumps are similar (impaired QS response), the underlying mechanisms are different. This ‘apparent redundancy' of MDR efflux systems can be understood as a P. aeruginosa strategy to keep the robustness of the QS regulatory network and modulate its output in response to different signals

Design of Quorum Sensing Inhibitor−Polymer Conjugates to Penetrate Pseudomonas aeruginosa Biofilms

Antimicrobial resistance (AMR) is a global threat to public health with a forecast of a negative financial impact of one trillion dollars per annum, hence novel therapeutics are urgently needed. The resistance of many bacteria against current drugs is further augmented by the ability of these microbes to form biofilms where cells are encased in a slimy extracellular matrix and either adhered to a surface or forming cell aggregates. Biofilms form physiochemical barriers against the penetration of treatments such as small molecule antibacterials, rendering most treatments ineffective. Pseudomonas aeruginosa, a priority pathogen of immediate concern, controls biofilm formation through multiple layers of gene regulation pathways including quorum sensing (QS), a cell-to-cell signaling system. We have recently reported a series of inhibitors of the PqsR QS regulator from this organism that can potentiate the action of antibiotics. However, these QS inhibitors (QSIs) have shown modest effects on biofilms in contrast with planktonic cultures due to poor penetration through the biofilm matrix. To enhance the delivery of the inhibitors, a small library of polymers was designed as carriers of a specific QSI, with variations in the side chains to introduce either positively charged or neutral moieties to aid penetration into and through the P. aeruginosa biofilm. The synthesized polymers were evaluated in a series of assays to establish their effects on the inhibition of the Pqs QS system in P. aeruginosa, the levels of inhibitor release from polymers, and their impact on biofilm formation. A selected cationic polymer–QSI conjugate was found to penetrate effectively through biofilm layers and to release the QSI. When used in combination with ciprofloxacin, it enhanced the biofilm antimicrobial activity of this antibiotic compared to free QSI and ciprofloxacin under the same conditions

Growth rate and nutrient limitation as key drivers of extracellular quorum sensing signal molecule accumulation in Pseudomonas aeruginosa

In Pseudomonas aeruginosa, quorum sensing (QS) depends on an interconnected regulatory hierarchy involving the Las, Rhl and Pqs systems, which are collectively responsible for the co-ordinated synthesis of a diverse repertoire of N-acylhomoserine lactones (AHLs) and 2-alkyl-4-quinolones (AQs). Apparent population density-dependent phenomena such as QS may, however, be due to growth rate and/or nutrient exhaustion in batch culture. Using continuous culture, we show that growth rate and population density independently modulate the accumulation of AHLs and AQs such that the highest concentrations are observed at a slow growth rate and high population density. Carbon source (notably succinate), nutrient limitation (C, N, Fe, Mg) or growth at 25 °C generally reduces AHL and AQ levels, except for P and S limitation, which result in substantially higher concentrations of AQs, particularly AQ N-oxides, despite the lower population densities achieved. Principal component analysis indicates that ~26 % variation is due to nutrient limitation and a further 30 % is due to growth rate. The formation of N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) turnover products such as the ring opened form and tetramic acid varies with the limiting nutrient limitation and anaerobiosis. Differential ratios of N-butanoyl-homoserine lactone (C4-HSL), 3OC12-HSL and the AQs as a function of growth environment are clearly apparent. Inactivation of QS by mutation of three key genes required for QS signal synthesis (lasI, rhlI and pqsA) substantially increases the concentrations of key substrates from the activated methyl cycle and aromatic amino acid biosynthesis, as well as ATP levels, highlighting the energetic drain that AHL and AQ synthesis and hence QS impose on P. aeruginosa