Rapid Screening of Quorum-Sensing Signal N-Acyl Homoserine Lactones by an In Vitro Cell-Free Assay

ABSTRACT A simple, sensitive, and rapid cell-free assay system was developed for detection of N-acyl homoserine lactone (AHL) autoinducers involved in bacterial quorum sensing (QS). The present approach improves upon previous whole-cell biosensor-based approaches in its utilization of a cell-free assay approach to conduct bioassays. The cell-free assay was derived from the AHL biosensor bacterium Agrobacterium tumefaciens NTL4(pCF218)(pCF372), allowing the expression of β-galactosidase upon addition of exogenous AHLs. We have shown that β-galactosidase expression is possible in cell-free solution [lysate from Agrobacterium tumefaciens NTL4(pCF218)(pCF372) culture]. Assay detection limits with the use of chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) ranged from approximately 100 nM to 300 nM depending on the specific AHL. Replacement (of X-Gal) with the luminescent substrate Beta-Glo increased sensitivity to AHLs by 10-fold. A major advantage of the cell-free assay system is elimination of time-consuming steps for biosensor cell culture conditioning, which are required prior to whole-cell bioassays. This significantly reduced assay times from greater than 24 h to less than 3 h, while maintaining high sensitivity. Assay lysate may be prepared in bulk and stored (−80°C) over 6 months for future use. Finally, the present protocol may be adapted for use with other biosensor strains and be used in high-throughput AHL screening of bacteria or metagenomic libraries.

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Priming winter wheat seeds with the bacterial quorum sensing signal N-hexanoyl-L-homoserine lactone (C6-HSL) shows potential to improve plant growth and seed yield

10.1101/490391 ◽

2018 ◽

Author(s):

Olena Moshynets ◽

Lidia M. Babenko ◽

Sergiy P. Rogalsky ◽

Olga S. Iungin ◽

Jessica Foster ◽

...

Keyword(s):

Quorum Sensing ◽

Winter Wheat ◽

Seed Priming ◽

Homoserine Lactone ◽

Field Trials ◽

Growth And Yield ◽

Cereal Crop ◽

Bacterial Quorum Sensing ◽

Bacterial Quorum

Several model plants are known to respond to bacterial quorum sensing molecules with altered root growth and gene expression patterns and induced resistance to plant pathogens. These compounds may represent novel elicitors that could be applied as seed primers to enhance cereal crop resistance to pathogens and abiotic stress and to improve yields. We investigated whether the acyl-homoserine lactone N-hexanoyl-L-homoserine lactone (C6-HSL) impacted winter wheat (Triticum aestivum L.) seed germination, plant development and productivity, using two Ukrainian varieties, Volodarka and Yatran 60, in both in vitro experiments and field trials. In vitro germination experiments indicated that C6-HSL seed priming had a small but significant positive impact on germination levels (1.2x increase, p < 0.0001), coleoptile and radicle development (1.4x increase, p < 0.0001). Field trials over two growing seasons (2015-16 and 2016-17) also demonstrated significant improvements in biomass at the tillering stage (1.4x increase, p < 0.0001), and crop structure and productivity at maturity including grain yield (1.4 – 1.5x increase, p < 0.0007) and quality (1.3x increase in good grain, p < 0.0001). In some cases variety effects were observed (p ≤ 0.05) suggesting that the effect of C6-HSL seed priming might depend on plant genetics, and some benefits of priming were also evident in F1 plants grown from seeds collected the previous season (p ≤ 0.05). These field-scale findings suggest that bacterial acyl-homoserine lactones such as C6-HSL could be used to improve cereal crop growth and yield and reduce reliance on fungicides and fertilisers to combat pathogens and stress.

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Influence of the luxR Regulatory Gene Dosage and Expression Level on the Sensitivity of the Whole-Cell Biosensor to Acyl-Homoserine Lactone

Biosensors ◽

10.3390/bios11060166 ◽

2021 ◽

Vol 11 (6) ◽

pp. 166

Author(s):

Sergey Bazhenov ◽

Uliana Novoyatlova ◽

Ekaterina Scheglova ◽

Vadim Fomin ◽

Svetlana Khrulnova ◽

...

Keyword(s):

Gene Dosage ◽

Regulatory Gene ◽

Homoserine Lactone ◽

Threshold Concentration ◽

Expression Level ◽

Whole Cell ◽

Lux Biosensors ◽

Order Of Magnitude ◽

Whole Cell Biosensor ◽

Cell Biosensor

Aliivibrio fischeri LuxR and Aliivibrio logei LuxR1 and LuxR2 regulatory proteins are quorum sensing transcriptional (QS) activators, inducing promoters of luxICDABEG genes in the presence of an autoinducer (3-oxo-hexanoyl-l-homoserine lactone). In the Aliivibrio cells, luxR genes are regulated by HNS, CRP, LitR, etc. Here we investigated the role of the luxR expression level in LuxI/R QS system functionality and improved the whole-cell biosensor for autoinducer detection. Escherichia coli-based bacterial lux-biosensors were used, in which Photorhabdus luminescensluxCDABE genes were controlled by LuxR-dependent promoters and luxR, luxR1, or luxR2 regulatory genes. We varied either the dosage of the regulatory gene in the cells using additional plasmids, or the level of the regulatory gene expression using the lactose operon promoter. It was shown that an increase in expression level, as well as dosage of the regulatory gene in biosensor cells, leads to an increase in sensitivity (the threshold concentration of AI is reduced by one order of magnitude) and to a two to threefold reduction in response time. The best parameters were obtained for a biosensor with an increased dosage of luxRA. fischeri (sensitivity to 3-oxo-hexanoyl-l-homoserine lactone reached 30–100 pM).

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Host modification of a bacterial quorum-sensing signal induces a phenotypic switch in bacterial symbionts

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1706879114 ◽

2017 ◽

Vol 114 (40) ◽

pp. E8488-E8497 ◽

Cited By ~ 37

Author(s):

Cleo Pietschke ◽

Christian Treitz ◽

Sylvain Forêt ◽

Annika Schultze ◽

Sven Künzel ◽

...

Keyword(s):

Quorum Sensing ◽

Bacterial Communities ◽

Homoserine Lactone ◽

Innate Immune ◽

Expression Data ◽

Phenotypic Switch ◽

Host Colonization ◽

Bacterial Quorum Sensing ◽

Bacterial Quorum ◽

Quorum Sensing Signals

Bacterial communities colonize epithelial surfaces of most animals. Several factors, including the innate immune system, mucus composition, and diet, have been identified as determinants of host-associated bacterial communities. Here we show that the early branching metazoan Hydra is able to modify bacterial quorum-sensing signals. We identified a eukaryotic mechanism that enables Hydra to specifically modify long-chain 3-oxo-homoserine lactones into their 3-hydroxy-HSL counterparts. Expression data revealed that Hydra’s main bacterial colonizer, Curvibacter sp., responds differentially to N-(3-hydroxydodecanoyl)-l-homoserine lactone (3OHC12-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL). Investigating the impacts of the different N-acyl-HSLs on host colonization elucidated that 3OHC12-HSL allows and 3OC12-HSL represses host colonization of Curvibacter sp. These results show that an animal manipulates bacterial quorum-sensing signals and that this modification leads to a phenotypic switch in the bacterial colonizers. This mechanism may enable the host to manipulate the gene expression and thereby the behavior of its bacterial colonizers.

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The Bacterial Quorum-Sensing Signal MoleculeN-3-Oxo-Dodecanoyl-l-Homoserine Lactone Reciprocally Modulates Pro- and Anti-Inflammatory Cytokines in Activated Macrophages

The Journal of Immunology ◽

10.4049/jimmunol.1300368 ◽

2013 ◽

Vol 191 (1) ◽

pp. 337-344 ◽

Cited By ~ 36

Author(s):

Yifat Glucksam-Galnoy ◽

Roy Sananes ◽

Nava Silberstein ◽

Pnina Krief ◽

Vladimir V. Kravchenko ◽

...

Keyword(s):

Quorum Sensing ◽

Inflammatory Cytokines ◽

Homoserine Lactone ◽

Activated Macrophages ◽

Bacterial Quorum Sensing ◽

Anti Inflammatory ◽

Bacterial Quorum

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Non-native autoinducer analogs capable of modulating the SdiA quorum sensing receptor inSalmonella entericaserovar Typhimurium

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.14.243 ◽

2018 ◽

Vol 14 ◽

pp. 2651-2664 ◽

Cited By ~ 3

Author(s):

Matthew J Styles ◽

Helen E Blackwell

Keyword(s):

Quorum Sensing ◽

Small Molecules ◽

Homoserine Lactone ◽

Interspecies Interactions ◽

Host Colonization ◽

Serovar Typhimurium ◽

Coordinate Group ◽

A Cell ◽

Production Host

Quorum sensing (QS) allows many common bacterial pathogens to coordinate group behaviors such as virulence factor production, host colonization, and biofilm formation at high population densities. This cell–cell signaling process is regulated byN-acyl L-homoserine lactone (AHL) signals, or autoinducers, and LuxR-type receptors in Gram-negative bacteria. SdiA is an orphan LuxR-type receptor found inEscherichia, Salmonella, Klebsiella, and Enterobactergenera that responds to AHL signals produced by other species and regulates genes involved in several aspects of host colonization. The inhibition of QS using non-native small molecules that target LuxR-type receptors offers a non-biocidal approach for studying, and potentially controlling, virulence in these bacteria. To date, few studies have characterized the features of AHLs and other small molecules capable of SdiA agonism, and no SdiA antagonists have been reported. Herein, we report the screening of a set of AHL analogs to both uncover agonists and antagonists of SdiA and to start to delineate structure–activity relationships (SARs) for SdiA:AHL interactions. Using a cell-based reporter of SdiA inSalmonella entericaserovar Typhimurium, several non-natural SdiA agonists and the first set of SdiA antagonists were identified and characterized. These compounds represent new chemical probes for exploring the mechanisms by which SdiA functions during infection and its role in interspecies interactions. Moreover, as SdiA is highly stable when produced in vitro, these compounds could advance fundamental studies of LuxR-type receptor:ligand interactions that engender both agonism and antagonism.

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Priming winter wheat seeds with the bacterial quorum sensing signal N-hexanoyl-L-homoserine lactone (C6-HSL) shows potential to improve plant growth and seed yield

PLoS ONE ◽

10.1371/journal.pone.0209460 ◽

2019 ◽

Vol 14 (2) ◽

pp. e0209460 ◽

Cited By ~ 13

Author(s):

Olena V. Moshynets ◽

Lidia M. Babenko ◽

Sergiy P. Rogalsky ◽

Olga S. Iungin ◽

Jessica Foster ◽

...

Keyword(s):

Quorum Sensing ◽

Plant Growth ◽

Winter Wheat ◽

Seed Yield ◽

Homoserine Lactone ◽

Bacterial Quorum Sensing ◽

Bacterial Quorum ◽

Wheat Seeds

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Novel bacteria degrading N-acylhomoserine lactones and their use as quenchers of quorum-sensing-regulated functions of plant-pathogenic bacteria

Microbiology ◽

10.1099/mic.0.26375-0 ◽

2003 ◽

Vol 149 (8) ◽

pp. 1981-1989 ◽

Cited By ~ 164

Author(s):

Stéphane Uroz ◽

Cathy D'Angelo-Picard ◽

Aurélien Carlier ◽

Miena Elasri ◽

Carine Sicot ◽

...

Keyword(s):

Quorum Sensing ◽

Pathogenic Bacteria ◽

Rhodococcus Erythropolis ◽

Homoserine Lactone ◽

Signal Molecules ◽

In Planta ◽

Signal Molecule ◽

Violacein Production ◽

Regulated Functions

Bacteria degrading the quorum-sensing (QS) signal molecule N-hexanoylhomoserine lactone were isolated from a tobacco rhizosphere. Twenty-five isolates degrading this homoserine lactone fell into six groups according to their genomic REP-PCR and rrs PCR-RFLP profiles. Representative strains from each group were identified as members of the genera Pseudomonas, Comamonas, Variovorax and Rhodococcus. All these isolates degraded N-acylhomoserine lactones other than the hexanoic acid derivative, albeit with different specificity and kinetics. One of these isolates, Rhodococcus erythropolis strain W2, was used to quench QS-regulated functions of other microbes. In vitro, W2 strongly interfered with violacein production by Chromobacterium violaceum, and transfer of pathogenicity in Agrobacterium tumefaciens. In planta, R. erythropolis W2 markedly reduced the pathogenicity of Pectobacterium carotovorum subsp. carotovorum in potato tubers. These series of results reveal the diversity of the QS-interfering bacteria in the rhizosphere and demonstrate the validity of targeting QS signal molecules to control pathogens with natural bacterial isolates.

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Immunization with 3-oxododecanoyl-l-homoserine lactone–protein conjugate protects mice from lethal Pseudomonas aeruginosa lung infection

Journal of Medical Microbiology ◽

10.1099/jmm.0.46658-0 ◽

2006 ◽

Vol 55 (10) ◽

pp. 1381-1387 ◽

Cited By ~ 68

Author(s):

Shinichi Miyairi ◽

Kazuhiro Tateda ◽

Etsu T. Fuse ◽

Chihiro Ueda ◽

Hiroaki Saito ◽

...

Keyword(s):

Quorum Sensing ◽

Specific Antibody ◽

Bacterial Infections ◽

Inflammatory Responses ◽

Critical Role ◽

Lung Infection ◽

Homoserine Lactone ◽

Vaccine Strategy ◽

Protein Conjugate

Quorum-sensing systems have been reported to play a critical role in the pathogenesis of several bacterial infections. Recent data have demonstrated that Pseudomonas N-3-oxododecanoyl-l-homoserine lactone (3-oxo-C12-homoserine lactone, 3-oxo-C12-HSL), but not N-butanoyl-l-homoserine lactone (C4-HSL), induces apoptosis in macrophages and neutrophils. In the present study, the effects of active immunization with 3-oxo-C12-HSL–carrier protein conjugate on acute P. aeruginosa lung infection in mice were investigated. Immunization with 3-oxo-C12-HSL–BSA conjugate (subcutaneous, four times, at 2-week intervals) elaborated significant amounts of specific antibody in serum. Control and immunized mice were intranasally challenged with approximately 3×106 c.f.u. P. aeruginosa PAO1, and survival was then compared. All control mice died by day 2 post bacterial challenge, while 36 % of immunized mice survived to day 4 (P<0.05). Interestingly, bacterial numbers in the lungs did not differ between control and immunized groups, whereas the levels of pulmonary tumour necrosis factor (TNF)-α in the immunized mice were significantly lower than those of control mice (P<0.05). Furthermore, the extractable 3-oxo-C12-HSL levels in serum and lung homogenate were also significantly diminished in the immunized mice. Immune serum completely rescued reduction of cell viability by 3-oxo-C12-HSL-mediated apoptosis in macrophages in vitro. These results demonstrated that specific antibody to 3-oxo-C12-HSL plays a protective role in acute P. aeruginosa infection, probably through blocking of host inflammatory responses, without altering lung bacterial burden. The present data identify a promising potential vaccine strategy targeting bacterial quorum-sensing molecules, including autoinducers.

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