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Antimicrobial resistance (AMR) represents a critical global health crisis. An innovative strategy to deal with AMR is to interfere with biofilm formation and bacterial quorum sensing (QS). In this study, newly designed autoinducer-2 (AI-2)-inspired compounds in targeting biofilm-associated infections were evaluated for their ability to inhibit biofilm formation in Staphylococcus aureus and Pseudomonas aeruginosa . The most effective compounds, 5d , 5e , and 7b , exhibited potent antibiofilm activity with minimal inhibitory concentrations in the low microgram per mL range. Detailed biological assays confirmed that the antibiofilm activity was primarily driven through AI-2 QS inhibition rather than direct antimicrobial effects. The combination of different spectroscopic techniques, such as differential scanning fluorimetry, intrinsic tryptophan fluorescence, circular dichroism, and nuclear magnetic resonance, elucidated the binding between the compounds and the LsrK enzyme, a key player in AI-2 mediated QS. Our findings highlight the potential of these novel QS inhibitors as promising therapeutic agents against biofilm-associated infections.

Background: Imbalanced intestinal microbiota and damage to the intestinal barrier contribute to the development of necrotizing enterocolitis (NEC). Autoinducer-2 (AI-2) plays a crucial role in repairing intestinal damage and reducing inflammation., Objective: This study aimed to investigate the impact of AI-2 on the expression of intestinal zonula occludens-1 (ZO-1) and occludin proteins in NEC. We evaluated its effects in vivo using NEC mice and in vitro using lipopolysaccharide (LPS)-stimulated intestinal cells., Methods: Pathological changes in the intestines of neonatal mice were assessed using histological staining and scoring. Cell proliferation was measured using the cell counting kit-8 (CCK-8) assay to determine the optimal conditions for LPS and AI-2 interventions. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA levels of matrix metalloproteinase-3 (MMP3), protease activated receptor-2 (PAR2), interleukin-1β (IL-1β), and IL-6. Protein levels of MMP3, PAR2, ZO-1, and occludin were evaluated using western blot, immunohistochemistry, or immunofluorescence., Results: AI-2 alleviated NEC-induced intestinal damage (P < 0.05) and enhanced the proliferation of damaged IEC-6 cells (P < 0.05). AI-2 intervention reduced the mRNA and protein expressions of MMP3 and PAR2 in intestinal tissue and cells (P < 0.05). Additionally, it increased the protein levels of ZO-1 and occludin (P < 0.05), while reducing IL-1β and IL-6 mRNA expression (P < 0.05)., Conclusion: AI-2 intervention enhances the expression of tight junction proteins (ZO-1 and occludin), mitigates intestinal damage in NEC neonatal mice and IEC-6 cells, potentially by modulating PAR2 and MMP3 signaling. AI-2 holds promise as a protective intervention for NEC. AI-2 plays a crucial role in repairing intestinal damage and reducing inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Regulating quorum sensing (QS) signaling molecules could improve wastewater treatment but might increase antibiotic resistance. This study investigated the effects of exogenous C6-HSL on anaerobic sludge under oxytetracycline stress, with a focus on antibiotic resistance genes (ARGs) and the QS response. The results revealed that exogenous oxytetracycline increased the copy number of ARGs by more than 68.8 %. It also facilitated a 3.04-fold increase in the concentration of signaling molecules and increased the abundance of QS genes. Further addition of the C6-HSL accelerated oxytetracycline degradation, and reduced its residual concentration by 70.9 %, alleviating oxytetracycline stress on microbial communities, and correspondingly reducing stress release from AHL by 75.4 %. Importantly, this did not exacerbate antibiotic resistance, with no significant difference (p > 0.05) in the ARG abundance. These findings may provide valuable insights into the relationship between QS process and antibiotic resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Pseudomonas aeruginosa (P. aeruginosa) biofilm formation is a crucial cause of enhanced antibiotic resistance. Quorum sensing (QS) is involved in regulating biofilm formation; QS inhibitors block the QS signaling pathway as a new strategy to address bacterial resistance. This study investigated the potential and mechanism of L-HSL (N-(3-cyclic butyrolactone)-4-trifluorophenylacetamide) as a QS inhibitor for P. aeruginosa. The results showed that L-HSL effectively inhibited the biofilm formation and dispersed the pre-formed biofilm of P. aeruginosa. The production of extracellular polysaccharides and the motility ability of P. aeruginosa were suppressed by L-HSL. C. elegans infection experiment showed that L-HSL was non-toxic and provided protection to C. elegans against P. aeruginosa infection. Transcriptomic analysis revealed that L-HSL downregulated genes related to QS pathways and biofilm formation. L-HSL exhibits a promising potential as a therapeutic drug for P. aeruginosa infection. KEY POINTS: • Chemical synthesis of N-(3-cyclic butyrolactone)-4-trifluorophenylacetamide, named L-HSL. • L-HSL does not generate survival pressure on the growth of P. aeruginosa and can inhibit the QS system. • KEGG enrichment analysis found that after L-HSL treatment, QS-related genes were downregulated., (© 2024. The Author(s).)

The activation of the angiopoietin (Angpt)-Tie system is linked to endothelial dysfunction during sepsis. Bacterial quorum-sensing molecules function as pathogen-associated molecular patterns. However, their impact on the endothelium and the Angpt-Tie system remains unclear. Therefore, this study investigated whether treatment with N-3-oxododecanoyl homoserine lactone (3OC12-HSL), a quorum-sensing molecule derived from Pseudomonas aeruginosa, impaired endothelial function in human umbilical vein endothelial cells. 3OC12-HSL treatment impaired tube formation even at sublethal concentrations, and immunocytochemistry analysis revealed that it seemed to reduce vascular endothelial-cadherin expression at the cell-cell interface. Upon assessing the mRNA expression patterns of genes associated with the Angpt-Tie axis, the expressions of Angpt2, Forkhead box protein O1, Tie1, and vascular endothelial growth factor 2 were found to be upregulated in the 3OC12-HSL-treated cells. Moreover, western blot analysis revealed that 3OC12-HSL treatment increased Angpt2 expression. A co-immunoprecipitation assay was conducted to assess the effect of 3OC12-HSL on the IQ motif containing GTPase activating protein 1 (IQGAP1) and Rac1 complex and the interaction between these proteins was consistently maintained regardless of 3OC12-HSL treatment. Next, recombinant human (rh)-Angpt1 was added to assess whether it modulated the effects of 3OC12-HSL treatment. rh-Angpt1 addition increased cellular viability, improved endothelial function, and reversed the overall patterns of mRNA and protein expression in endothelial cells treated with 3OC12-HSL. Additionally, it was related to the increased expression of phospho-Akt and the IQGAP1 and Rac1 complex. Collectively, our findings indicated that 3OC12-HSL from Pseudomonas aeruginosa can impair endothelial integrity via the activation of the Angpt-Tie axis, which appeared to be reversed by rh-Angpt1 treatment., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: The formation of bacterial biofilm regulated by quorum sensing (QS) is a critical factor that contributes to infections of indwelling medical devices. Autoinducer-2 (AI-2), as a signal molecule in QS, plays a crucial role in mediating bacterial signaling and regulating their biological behavior. This study investigated the impact of ultrasonic vibration at varying frequencies on biofilm formation in a mixture of Staphylococcus aureus and Escherichia coli., Methods: By exciting ultrasound at different frequencies (20, 100 and 200 kHz), a vibration with an amplitude of 100 nm was generated on the material surface located at the bottom of the petri dish containing mixed bacteria. We measured the content of AI-2 and bacteria in the mixed bacterial solution and bioburden on material surfaces at different time points during culture. In addition, the relationships among AI-2 content, bacterial concentration and distribution were assessed through finite-element analysis of acoustic streaming under ultrasonic vibration., Results: The AI-2 gradient is influenced by the diversity of acoustic streaming patterns on the material surface and in the mixed bacterial solution caused by ultrasonic vibration at different frequencies, thereby regulating biofilm formation. The experimental results showed that the optimal inhibition effect on AI-2 and minimal bacterial adhesion degree was achieved when applying an ultrasonic frequency of 100 kHz with a power intensity of 46.1 mW/cm 2 under an amplitude of 100 nm., Conclusion: Ultrasound can affect the QS system of bacteria, leading to alterations in their biological behavior. Different species of bacteria exhibit varying degrees of chemotaxis toward different frequencies., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

The host transcriptional activator Early growth response 1 (EGR1) plays a vital role in cell cycle and differentiation, cell proliferation, and regulation of cytokines and several growth factors. It is an immediate-early gene that is expressed as an initial response to various environmental stimuli. Bacterial infection is one such factor that can trigger the expression of EGR1 in host. Therefore, it is imperative to understand expression of EGR1 during early stages of host-pathogen interaction. Streptococcus pyogenes is an opportunistic bacteria causing skin and respiratory tract infections in humans. The quorum-sensing molecule, N-(3-oxododecanoyl)-l-homoserine lactone (Oxo-C12), not synthesised by S. pyogenes, can be sensed by S. pyogenes leading to molecular changes in the pathogen. In this study, we investigated the role of Oxo-C12 on EGR1 regulation in lung epithelial and murine macrophage cell line upon S. pyogenes infection. We report that Oxo-C12 sensitised S. pyogenes upregulates the transcriptional expression of EGR1 through ERK1/2 pathway. It was observed that EGR1 was not involved in the intial attachment of S. pyogenes to A549 cells. However, inhibition of EGR1 in macrophage cell line, J774A.1, through the ERK1/2 pathway resulted in decreased adhesion of S. pyogenes. The EGR1 upregulation by Oxo-C12 sensitised S. pyogenes plays a vital role in enhancing the survival of S. pyogenes in murine macrophages, leading to persistent infection. Thus, understanding the molecular modulation in the host during bacterial infection will further help develop therapeutics to target specific sites., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The limited diversity in targets of available antibiotic therapies has put tremendous pressure on the treatment of bacterial pathogens, where numerous resistance mechanisms that counteract their function are becoming increasingly prevalent. Here, we utilize an unconventional anti-virulence screen of host-guest interacting macrocycles, and identify a water-soluble synthetic macrocycle, Pillar[5]arene, that is non-bactericidal/bacteriostatic and has a mechanism of action that involves binding to both homoserine lactones and lipopolysaccharides, key virulence factors in Gram-negative pathogens. Pillar[5]arene is active against Top Priority carbapenem- and third/fourth-generation cephalosporin-resistant Pseudomonas aeruginosa and Acinetobacter baumannii, suppressing toxins and biofilms and increasing the penetration and efficacy of standard-of-care antibiotics in combined administrations. The binding of homoserine lactones and lipopolysaccharides also sequesters their direct effects as toxins on eukaryotic membranes, neutralizing key tools that promote bacterial colonization and impede immune defenses, both in vitro and in vivo. Pillar[5]arene evades both existing antibiotic resistance mechanisms, as well as the build-up of rapid tolerance/resistance. The versatility of macrocyclic host-guest chemistry provides ample strategies for tailored targeting of virulence in a wide range of Gram-negative infectious diseases., (© 2023. The Author(s).)

Quorum Sensing allows bacteria to sense their population density via diffusible N-acyl homoserine lactone (N-HL) signaling molecules. Upon reaching a high enough cell density, bacteria will collectively exhibit a phenotype. Until recently, methods used for detection of N-HLs have not considered the chirality of these molecules and it was assumed that only the L-enantiomer was produced by bacteria. The production and effects of D-N-HLs have rarely been studied. In this work, the temporal production of D-N-HLs by the plant pathogen Pectobacterium atrosepticum and the human pathogen Pseudomonas aeruginosa are reported. Both bacteria produced D-N-HLs in significant amounts and in some cases their concentrations were higher than other low abundance L-N-HLs. Previously unreported D-enantiomers of N-3-oxoacyl and N-3-hydroxyacyl homoserine lactones were detected in P. atrosepticum. Interestingly, L-N-HLs produced in the lowest concentrations had relatively higher amounts of their corresponding D-enantiomers. Potential sources of D-N-HLs and their significance are considered., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Dhaubhadel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Streptococcus suis LuxS/AI-2 quorum sensing system regulates biofilm formation, resulting in increased pathogenicity and drug resistance, and diminished efficacy of antibiotic treatment. The remaining peony seed cake after oil extraction is rich in monoterpenoid glycosides, which can inhibit the formation of bacterial biofilm. In this study, we investigated the effect of seven major monocomponents (suffruticosol A, suffruticosol B, suffruticosol C, paeonifloin, albiflorin, trans-ε-viniferin, gnetin H) of peony seed meal on minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of S. suis . The results showed that the MICs of the seven single components were all greater than 200 μg/mL, with no significant bacteriostatic and bactericidal advantages. Crystal violet staining and scanning electron microscope observation showed that the seven single components had a certain inhibitory effect on the biofilm formation ability of S. suis at sub-MIC concentration. Among them, the ability of paeoniflorin to inhibit biofilm was significantly higher than that of the other six single components. AI-2 signaling molecules were detected by bioreporter strain Vibrio harvey BB170 . The detection results of AI-2 signal molecules found that at 1/2 MIC concentration, paeoniflorin significantly inhibited the production of S. suis AI-2 signal, and the inhibitory effect was better than that of the other six single components. In addition, molecular docking analysis revealed that paeoniflorin had a significant binding activity with LuxS protein compared with the other six single components. The present study provides evidence that paeoniflorin plays a key role in the regulation of the inhibition of S. suis LuxS/AI-2 system and biofilm formation in peony seed meal., Competing Interests: The authors declare no conflict of interest.

The opportunistic bacterium Pseudomonas aeruginosa secretes the quorum-sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (C12) to co-ordinate gene expression profiles favorable for infection. Recent studies have demonstrated that high concentrations of C12 impair many aspects of host cell physiology, including mitochondrial function and cell viability. The cytotoxic effects of C12 are mediated by the lactonase enzyme, Paraoxonase 2 (PON2), which hydrolyzes C12 to a reactive metabolite. However, the influence of C12 on host cell physiology at concentrations observed in patients infected with P. aeruginosa is largely unknown. Since the primary site of P. aeruginosa infections is the mammalian airway, we sought to investigate how PON2 modulates the effects of C12 at subtoxic concentrations using immortalized murine tracheal epithelial cells (TECs) isolated from wild-type (WT) or PON2-knockout (PON2-KO) mice. Our data reveal that C12 at subtoxic concentrations disrupts mitochondrial bioenergetics to hinder cellular proliferation in TECs expressing PON2. Subtoxic concentrations of C12 disrupt normal mitochondrial network morphology in a PON2-dependent manner without affecting mitochondrial membrane potential. In contrast, higher concentrations of C12 depolarize mitochondrial membrane potential and subsequently trigger caspase signaling and apoptotic cell death. These findings demonstrate that different concentrations of C12 impact distinct aspects of host airway epithelial cell physiology through PON2 activity in mitochondria., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

There is an urgent need to validate new drug targets and identify small molecules that possess activity against both drug-resistant and drug-sensitive bacteria. The enzymes belonging to amino acid biosynthesis have been shown to be essential for growth in vitro, in vivo and have not been exploited much for the development of anti-tubercular agents. Here, we have identified small molecule inhibitors targeting homoserine acetyl transferase (HSAT, MetX, Rv3341) from M. tuberculosis. MetX catalyses the first committed step in L-methionine and S-adenosyl methionine biosynthesis resulting in the formation of O-acetyl-homoserine. Using CRISPRi approach, we demonstrate that conditional repression of metX resulted in inhibition of M. tuberculosis growth in vitro. We have determined steady state kinetic parameters for the acetylation of L-homoserine by Rv3341. We show that the recombinant enzyme followed Michaelis-Menten kinetics and utilizes both acetyl-CoA and propionyl-CoA as acyl-donors. High-throughput screening of a 2443 compound library resulted in identification of small molecule inhibitors against MetX enzyme from M. tuberculosis. The identified lead compounds inhibited Rv3341 enzymatic activity in a dose dependent manner and were also active against HSAT homolog from S. aureus. Molecular docking of the identified primary hits predicted residues that are essential for their binding in HSAT homologs from M. tuberculosis and S. aureus. ThermoFluor assay demonstrated direct binding of the identified primary hits with HSAT proteins. Few of the identified small molecules were able to inhibit growth of M. tuberculosis and S. aureus in liquid cultures. Taken together, our findings validated HSAT as an attractive target for development of new broad-spectrum anti-bacterial agents that should be effective against drug-resistant bacteria., (© 2022. The Author(s).)

Boron is an essential element for autoinducer-2 (AI-2) synthesis of quorum sensing (QS) system, which affects bacterial collective behavior. As a living biocatalyst, biofilms can stably catalyze the activity of intracellular enzymes. However, it is unclear how boron affects biofilm formation in E. coli, particularly recombinant E. coli with intracellular enzymes. This study screened different boron derivatives to explore their effect on biofilm formation. The stress response of biofilm formation to boron was illuminated by analyzing AI-2 activity, extracellular polymeric substances (EPS) composition, gene expression levels, etc. Results showed that boron derivatives promote AI-2 activity in QS system. After treatment with H3BO3 (0.6 mM), the AI-2 activity increased by 65.99%, while boron derivatives increased the biomass biofilms in the order H3BO3 > NaBO2 > Na2B4O7 > NaBO3. Moreover, treatment with H3BO3 (0.6 mM) increased biomass by 88.54%. Meanwhile, AI-2 activity had a linear correlation with polysaccharides and protein of EPS at 0−0.6 mM H3BO3 and NaBO2 (R2 > 0.8). Furthermore, H3BO3 upregulated the expression levels of biofilm formation genes, quorum sensing genes, and flagellar movement genes. These findings demonstrated that boron promoted biofilm formation by upregulating the expression levels of biofilm-related genes, improving the QS system AI-2 activity, and increasing EPS secretion in E. coli.

We studied the effect of early accumulation of N-3-oxo-dodecanoyl-homoserine lactone on the suppression of Pseudomonas aeruginosa reproduction, biofilm formation, and elastase activity. N-3-oxo-dodecanoyl-homoserine lactone in various concentrations was added to the P. aeruginosa culture, and changes in the concentration of bacteria and the formation of biofilms were studied in dynamics. N-3-oxo-dodecanoyl-homoserine lactone in a concentration of 25 μM, decelerated proliferation of bacterial cells during the first 6 h of culturing (p<0.05) and stimulated biofilm formation after 18 h of culturing. Elastase activity of P. aeruginosa increased significantly after addition of N-3-oxo-dodecanoyl-homoserine lactone in a concentration of 0.75 μM., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Microbes employ autoinducers of quorum sensing (QS) for population communication. Although the autoinducer of Pseudomonas aeruginosa LasI-LasR system, N-(3-oxododecanoyl)- l-homoserine lactone (3OC12), has been reported with deleterious effects on host cells, its biological effects on integrity of the intestinal epithelium and epithelial barrier are still unclear and need further investigation. In the present study, flow cytometry, transcriptome analysis and western blot technology have been adopted to investigate the potential molecular mechanisms of 3OC12 and its structurally similar analogs damage to intestinal epithelial cells. Our results indicated that 3OC12 and 3OC14 trigger apoptosis rather than necrosis and ferroptosis in intestinal epithelial cells. RNA-sequencing combined with bioinformatics analysis showed that 3OC12 and 3OC14 reduced the expression of genes from extracellular matrix (ECM)-receptor interaction pathway. Consistently, protein expressions from ECM and tight junction-associated pathway were significantly reduced after 3OC12 and 3OC14 challenge. In addition, 3OC12 and 3OC14 led to blocked cell cycle, decreased mitochondrial membrane potential, increased reactive oxygen species level and elevated Ca 2+ concentration. Reversely, the antioxidant NAC could effectively mitigate the reduced expression of ECM and tight junction proteins caused by 3OC12 and 3OC14 challenge. Collectively, this study demonstrated that QS autoinducer exposure to intestinal epithelial cells ablates the ECM and tight junctions by triggering oxidative stress and apoptosis, and finally disrupts the intestinal epithelial barrier. These findings provide a rationale for defensing QS-dependent bacterial infections and potential role of NAC for alleviating the syndrome., (© 2020 Wiley Periodicals LLC.)

Quorum sensing of Acinetobacter nosocomialis for cell-to-cell communication produces N -3-hydroxy dodecanoyl-DL-homoserine lactone (OH-dDHL) by an AnoR/I two-component system. However, OH-dDHL-driven apoptotic mechanisms in hosts have not been clearly defined. Here, we investigated the induction of apoptosis signaling pathways in bone marrow-derived macrophages treated with synthetic OH-dDHL. Moreover, the quorum-sensing system for virulence regulation was evaluated in vivo using wild-type and anoI -deletion mutant strains. OH-dDHL decreased the viability of macrophage and epithelial cells in dose- and time-dependent manners. OH-dDHL induced Ca 2+ efflux and caspase-12 activation by ER stress transmembrane protein (IRE1 and ATF6a p50) aggregation and induced mitochondrial dysfunction through reactive oxygen species (ROS) production, which caused cytochrome c to leak. Pretreatment with a pan-caspase inhibitor reduced caspase-3, -8, and -9, which were activated by OH-dDHL. Pro-inflammatory cytokine and paraoxonase-2 (PON2) gene expression were increased by OH-dDHL. We showed that the anoI -deletion mutant strains have less intracellular invasion compared to the wild-type strain, and their virulence, such as colonization and dissemination, was decreased in vivo. Consequently, these findings revealed that OH-dDHL, as a virulence factor, contributes to bacterial infection and survival as well as the modification of host responses in the early stages of infection.

Sulfate-reducing bacteria (SRB) are culprits for microbiologically influenced corrosion, and biofilms are believed to play essential roles in the corrosion induced by SRB. However, little is known about the regulation of SRB biofilms. Quorum sensing signal molecules acyl-homoserine lactones (AHLs) and autoinducer-2 (AI-2) regulate biofilm formation of many bacteria. In this study, the production of AHLs and AI-2 by one SRB strain, Desulfovibrio sp. Huiquan2017, was detected, and the effect of exogenous AI-2 on bacterial biofilm formation was discussed. It was found that the cell-free supernatants of Desulfovibrio sp. Huiquan2017 induced luminescence in a ∆luxS mutant strain Vibrio harveyi BB170, indicating the production of functional AI-2 by the bacterium. In the presence of exogenous AI-2, the growth of Desulfovibrio sp. Huiquan2017 and early biofilm formation were not affected, but the later stage of biofilm development was inhibited significantly. The biofilms became looser, smaller, and thinner, and contained less bacteria and extracellular polymeric substances (EPS). The inhibition effect of AI-2 on the biofilm development of Desulfovibrio sp. Huiquan2017 was mainly achieved through reducing the amount of EPS in biofilms. These findings shed light on the biofilm regulation of SRB.

Bitter receptors function primarily in sensing taste, but may also have other functions, such as detecting pathogenic organisms due to their agile response to foreign objects. The mouse taste receptor type-2 member 138 (TAS2R138) is a member of the G-protein-coupled bitter receptor family, which is not only found in the tongue and nasal cavity, but also widely distributed in other organs, such as the respiratory tract, gut, and lungs. Despite its diverse functions, the role of TAS2R138 in host defense against bacterial infection is largely unknown. Here, we show that TAS2R138 facilitates the degradation of lipid droplets (LDs) in neutrophils during Pseudomonas aeruginosa infection through competitive binding with PPARG (peroxisome proliferator-activated receptor gamma) antagonist: N-(3-oxododecanoyl)-L-homoserine lactone (AHL-12), which coincidently is a virulence-bound signal produced by this bacterium (P. aeruginosa). The released PPARG then migrates from nuclei to the cytoplasm to accelerate the degradation of LDs by binding PLIN2 (perilipin-2). Subsequently, the TAS2R138-AHL-12 complex targets LDs to augment their degradation, and thereby facilitating the clearance of AHL-12 in neutrophils to maintain homeostasis in the local environment. These findings reveal a crucial role for TAS2R138 in neutrophil-mediated host immunity against P. aeruginosa infection.

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N 2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N 2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

The quorum-sensing (QS) signaling-dependent extracellular virulence factors of Pseudomonas aeruginosa can cause infections such as P. aeruginosa keratitis. P. aeruginosa communicates by secreting and sensing small chemical molecules called autoinducers in QS system. The key QS signal molecule, N -3-oxododecanoyl-homoserine lactone (3OC12HSL), can affect the behavior of host cells and initiate immune response. In this report we investigated the influence of 3OC12HSL on human corneal epithelial cells (HCECs) and the mechanisms of 3OC12HSL on activated toll-like receptor 2 (TLR2)-dependent interleukin-8 (IL-8) secretion in HCECs. Cells were cultured under different concentrations of 3OC12HSL. Cell viability was assessed using Crystal violet staining and the cell counting kit-8 assay. We demonstrated the administration of 3OC12HSL decreased HCEC viability and survival in a concentration- and time-dependent manner. At high concentrations, 3OC12HSL rapidly promoted a time-dependent increase in the expressions of TLR2 and TLR4. It was found that the nuclear translocation and expression of nuclear factor-κB (NF-κB) were also increased in response to 3OC12HSL treatment. The significantly elevated expressions of TLR2, TLR4, and NF-κB, encouraged us to further test their mechanisms that cause inflammatory response. Among the inflammatory factors examined (IL-6, IL-8, IL-10, and TNF-α), we found that IL-8 was significantly increased after treatment with 3OC12HSL and its expression was inhibited when TLR2 was specifically blocked or silenced. These results indicated that the QS signaling molecule 3OC12HSL could be recognized by the host innate immune system in HCECs. This recognition then triggered an immune inflammatory response involving the activation of TLR2 and an increase in expression of IL-8. This crosstalk between 3OC12HSL and host immunity in HCECs contributes to the development and progression of P. aeruginosa keratitis.

A new synthetic route for the quorum sensing signal Autoinducer-2 (AI-2) is described and used for the preparation of [4- 13 C]-AI-2 starting from [1- 13 C]-bromoacetic acid. The key step in this process was the enantioselective reduction of an intermediate ketone. This synthesis provides, selectively, both enantiomers of the labelled or unlabelled parent compound, ( R ) or ( S )-4,5-dihydroxypentane-2,3-dione (DPD) and was used for an improved synthesis of [1- 13 C]-AI-2.

N -3-(oxododecanoyl)-L-homoserine lactone (3-O-C12-HSL), a small bacterial signaling molecule secreted by Pseudomonas aeruginosa (P. aeruginosa) , can block dendritic cell (DC) maturation and participate in immune escape, but the underlying mechanism is unclear. We speculate that regulation of DC maturation and function by lncRNAs may be the mechanism by which 3-O-C12-HSL inhibits the immune response. We found that 3-O-C12-HSL increased the expression level of the lncRNA NRIR, impeding monocyte-derived dendritic cell (Mo-DC) maturation. In addition, we observed the effect of NRIR on the expression of CD40, CD80, HLA-DR and IL-6. NRIR overexpression significantly reduced the expression of Mo-DC surface markers, while 3-OC12-HSL did not significantly reduce the expression of Mo-DC surface markers after NRIR knockdown. These results indicate that 3-O-C12-HSL indeed affects the differentiation and maturation of Mo-DCs through NRIR. IL-6 stimulates T cell proliferation and activation, and we found that high NRIR expression reduced IL6 levels. However, under NRIR knockdown, 3-O-C12-HSL did not decrease IL-6 expression, suggesting that 3-O-C12-HSL may affect T cell activation through NRIR. This study is the first to elucidate the important role of a lncRNA in the mechanism of 3-O-C12-HSL activity. It also provides new ideas regarding P. aeruginosa infection pathogenesis.

The present study aimed to evaluate the anti-quorum sensing (anti-QS) and anti-proteolytic potentials of tarragon essential oil (TEO) and its major compounds against food-associated Pseudomonas spp. The activities were verified by in vitro, in silico and in situ approaches. In this work, methyl eugenol (ME)- and β-phellandrene (β-PH)-rich TEO was investigated. TEO at subMIC increased the percentage of saturated fatty acids in the bacterial membranes (from 7 to 22%) and exhibited anti-quorum sensing via decreasing the efficiency of QS autoinducer synthesis [3-oxo-C12-HSL (from 2.028 μg/mL to

The existence of crosstalk between quorum sensing systems limits their application in a complex environment. In this study, two completely orthogonal quorum sensing systems with self-produced autoinducers were built in one cell to enable the systems to be signal orthogonal and promoter orthogonal to each other. The systems were designed on the basis of the las system from Pseudomonas aeruginosa and the tra system from Agrobacterium tumefaciens . Both were optimized with respect to the orthogonality of signals and promoters by using a series of synthetic biology strategies and high-throughput screening. The systems were applied intracellularly, and an automatic delayed cascade circuit was successfully demonstrated, which can realize sequential gene expression without exogenous inducer. This circuit provides a new tool for biotechnological applications, such as metabolic regulation, that require sequential gene control. This cascade model expands the toolkit of synthetic biology research and indicates a high application potential of quorum sensing systems that are orthogonal to each other.

Background : The immune-inducing effect of the quorum sensing (QS) molecule autoinducer-2 (AI-2) on macrophages has not been previously comprehensively studied. Methods : We performed proteomic analysis on macrophages cocultured with purified Fusobacterium nucleatum ( F. nucleatum ) AI-2 and performed western blot analysis to verify the differential protein expression. We further used the Gene Expression Profiling Interactive Analysis and Tumor Immune Estimation Resource databases to analyze the expression of differentially expressed proteins in microbial-associated digestive tract tumors. Results : Based on proteomic analysis, we identified 46 upregulated proteins and 11 downregulated proteins. The upregulated proteins were mostly inflammatory factors such as tumor necrosis factor ligand superfamily member 9 (TNFSF9). These proteins have a range of biological functions associated with the regulation of inflammatory responses, apoptosis and tumorigenesis. TNFSF9 is highly expressed in pancreatic adenocarcinoma (PAAD) tissues and is associated with M1 polarization of macrophages. Conclusions : Our data indicated that F. nucleatum AI-2 induced inflammatory responses and activated multiple signaling pathways in macrophages. TNFSF9 is the most significantly differentially expressed protein induced by F. nucleatum AI-2 and is involved in regulating immune cell infiltration in PAAD. Thus, AI-2 may become a new focus for studying the relationship between bacteria and cancer.

Acyl-homoserine lactones (AHLs), are the key autoinducer molecules that mediate Pseudomonas aeruginosa associated quorum sensing. P. aeruginosa produces two types of AHLs; N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C 12 HSL) and N-butyryl-L-homoserine lactone (C4 HSL). AHLs are not only regulating the virulence gene of bacteria but also influence the host cell functions by interkingdom signaling. In this study, we explored the mechanism of AHLs induced calcium mobilization in human platelets. We found that 3-oxo-C 12 HSL but not C4 HSL induces intracellular calcium release. 3-oxo-C 12 HSL induced calcium mobilization was majorly contributed from the dense tubular system (DTS). Furthermore, 3-oxo-C 12 HSL also stimulates the store-operated Ca 2+ entry (SOCE) in platelet. Intracellular calcium rise was significantly lowered in rotenone, and bafilomycin pre-treated platelets suggesting partial involvement of mitochondria and acidic vacuoles. The significant effect of 3-oxo-C 12 HSL on calcium mobilization can alter the platelet functions that might results in thrombotic disorders in individuals infected with P. aeruginosa., (Copyright © 2019. Published by Elsevier Inc.)

Synthetic biology and metabolic engineering have expanded the possibilities for engineered cell-based systems. The addition of non-native biosynthetic and regulatory components can, however, overburden the reprogrammed cells. In order to avoid metabolic overload, an emerging area of focus is on engineering consortia, wherein cell subpopulations work together to carry out a desired function. This strategy requires regulation of the cell populations. Here, we design a synthetic co-culture controller consisting of cell-based signal translator and growth-controller modules that, when implemented, provide for autonomous regulation of the consortia composition. The system co-opts the orthogonal autoinducer AI-1 and AI-2 cell-cell signaling mechanisms of bacterial quorum sensing (QS) to enable cross-talk between strains and a QS signal-controlled growth rate controller to modulate relative population densities. We further develop a simple mathematical model that enables cell and system design for autonomous closed-loop control of population trajectories.

The pathogenicity of P. aeruginosa is dependent on quorum sensing (QS), an inter-bacterial communication system that can also modulate host biology. The innate immune function of the lung mucosal barrier is dependent on proper mitochondrial function. The purpose of this study was to define the mechanism by which bacterial factors modulate host lung epithelial cell mitochondrial function and to investigate novel therapies that ameliorate this effect. 3-oxo-C12-HSL disrupts mitochondrial morphology, attenuates mitochondrial bioenergetics, and induces mitochondrial DNA oxidative injury. Mechanistically, we show that 3-oxo-C12-HSL attenuates expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, antioxidant defense, and cellular respiration, and its downstream effectors in both BEAS-2B and primary lung epithelial cells. Overexpression of PGC-1α attenuates the inhibition in cellular respiration caused by 3-oxo-C12-HSL. Pharmacologic activation of PGC-1α restores barrier integrity in cells treated with 3-oxo-C12-HSL. These data demonstrate that the P. aeruginosa QS molecule, 3-oxo-C12-HSL, alters mitochondrial pathways critical for lung mucosal immunity. Genetic and pharmacologic strategies that activate the PGC-1α pathway enhance host epithelial cell mitochondrial function and improve the epithelial innate response to P. aeruginosa. Therapies that rescue PGC-1α function may provide a complementary approach in the treatment of P. aeruginosa infection.

The anaerobic gut pathogen, Clostridioides difficile, forms adherent biofilms that may play an important role in recurrent C. difficile infections. The mechanisms underlying C. difficile community formation and inter-bacterial interactions are nevertheless poorly understood. C. difficile produces AI-2, a quorum sensing molecule that modulates biofilm formation across many bacterial species. We found that a strain defective in LuxS, the enzyme that mediates AI-2 production, is defective in biofilm development in vitro. Transcriptomic analyses of biofilms formed by wild type (WT) and luxS mutant (luxS) strains revealed a downregulation of prophage loci in the luxS mutant biofilms compared to the WT. Detection of phages and eDNA within biofilms may suggest that DNA release by phage-mediated cell lysis contributes to C. difficile biofilm formation. In order to understand if LuxS mediates C. difficile crosstalk with other gut species, C. difficile interactions with a common gut bacterium, Bacteroides fragilis, were studied. We demonstrate that C. difficile growth is significantly reduced when co-cultured with B. fragilis in mixed biofilms. Interestingly, the absence of C. difficile LuxS alleviates the B. fragilis-mediated growth inhibition. Dual species RNA-sequencing analyses from single and mixed biofilms revealed differential modulation of distinct metabolic pathways for C. difficile WT, luxS and B. fragilis upon co-culture, indicating that AI-2 may be involved in induction of selective metabolic responses in B. fragilis. Overall, our data suggest that C. difficile LuxS/AI-2 utilises different mechanisms to mediate formation of single and mixed species communities.

Quorum sensing is a cell-cell communication mechanism mediated by chemical signals that leads to differential gene expression in response to high population density. Salmonella is unable to synthesize the autoinducer-1 (AI-1), N-acyl homoserine lactone (AHL), but is able to recognize AHLs produced by other microorganisms through SdiA protein. This study aimed to evaluate the fatty acid and protein profiles of Salmonella enterica serovar Enteritidis PT4 578 throughout time of cultivation in the presence of AHL. The presence of N-dodecanoyl-homoserine lactone (C12-HSL) altered the fatty acid and protein profiles of Salmonella cultivated during 4, 6, 7, 12 and 36 h in anaerobic condition. The profiles of Salmonella Enteritidis at logarithmic phase of growth (4 h of cultivation), in the presence of C12-HSL, were similar to those of cells at late stationary phase (36 h). In addition, there was less variation in both protein and fatty acid profiles along growth, suggesting that this quorum sensing signal anticipated a stationary phase response. The presence of C12-HSL increased the abundance of thiol related proteins such as Tpx, Q7CR42, Q8ZP25, YfgD, AhpC, NfsB, YdhD and TrxA, as well as the levels of free cellular thiol after 6 h of cultivation, suggesting that these cells have greater potential to resist oxidative stress. Additionally, the LuxS protein which synthesizes the AI-2 signaling molecule was differentially abundant in the presence of C12-HSL. The NfsB protein had its abundance increased in the presence of C12-HSL at all evaluated times, which is a suggestion that the cells may be susceptible to the action of nitrofurans or that AHLs present some toxicity. Overall, the presence of C12-HSL altered important pathways related to oxidative stress and stationary phase response in Salmonella., Competing Interests: The authors have declared that no competing interests exist.

Aims: The quorum-sensing molecule N‑(3‑oxododecanoyl)‑l‑homoserine lactone (C12-HSL), produced by the Gram negative human pathogenic bacterium Pseudomonas aeruginosa, modulates mammalian cell behavior. Our previous findings suggested that C12-HSL rapidly decreases viability and induces apoptosis in LS174T goblet cells., Main Methods: In this study, the effects of 100 μM C12-HSL on mitochondrial function and cell proliferation in LS174T cells treated for 4 h were evaluated by real-time PCR, enzyme-linked immunosorbent assay (ELISA) and flow cytometry., Key Findings: The results showed that the activities of mitochondrial respiratory chain complexes IV and V were significantly increased (P < 0.05) in LS174T cells after C12-HSL treatment, with elevated intracellular ATP generation (P < 0.05). Flow cytometry analysis revealed significantly increased intracellular Ca 2+ levels (P < 0.05), as well as disrupted mitochondrial activity and cell cycle arrest upon C12-HSL treatment. Apoptosis and cell proliferation related genes showed markedly altered expression levels (P < 0.05) in LS174T cells after C12-HSL treatment. Moreover, the paraoxonase 2 (PON2) inhibitor TQ416 (1 μM) remarkably reversed the above C12-HSL associated effects in LS174T cells., Significance: These findings indicated that C12-HSL alters mitochondrial energy production and function, and inhibits cell proliferation in LS174T cells, with PON2 involvement., (Copyright © 2018 Elsevier Inc. All rights reserved.)

β-Aminobutyric acid (BABA) induces broad-spectrum disease resistance, but also represses plant growth, which has limited its exploitation in crop protection. BABA perception relies on binding to the aspartyl-tRNA synthetase (AspRS) IBI1, which primes the enzyme for secondary defense activity. This study aimed to identify structural BABA analogues that induce resistance without stunting plant growth. Using site-directed mutagenesis, we demonstrate that the (l)-aspartic acid-binding domain of IBI1 is critical for BABA perception. Based on interaction models of this domain, we screened a small library of structural BABA analogues for growth repression and induced resistance against biotrophic Hyaloperonospora arabidopsidis (Hpa). A range of resistance-inducing compounds were identified, of which (R)-β-homoserine (RBH) was the most effective. Surprisingly, RBH acted through different pathways than BABA. RBH-induced resistance (RBH-IR) against Hpa functioned independently of salicylic acid, partially relied on camalexin, and was associated with augmented cell wall defense. RBH-IR against necrotrophic Plectosphaerella cucumerina acted via priming of ethylene and jasmonic acid defenses. RBH-IR was also effective in tomato against Botrytis cinerea. Metabolic profiling revealed that RBH, unlike BABA, does not majorly affect plant metabolism. RBH primes distinct defense pathways against biotrophic and necrotrophic pathogens without stunting plant growth, signifying strong potential for exploitation in crop protection., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

High-density lipoprotein (HDL) plays an important role in preventing atherosclerosis. The antioxidant effect of HDL is mostly associated with paraoxonase 1 (PON1) activity. Increasing PON1 activity using nutrients might improve HDL function and quality and thus, decrease atherosclerotic risk. We previously isolated and identified a novel active compound, lyso-DGTS (C20:5,0) from Nannochloropsis sp. ethanol extract. In the present study, its effect on PON1 activities was examined and the mechanism by which the compound affects PON1 activity was explored. Lyso-DGTS elevated recombinant PON1 (rePON1) lactonase and esterase activities in a dose- and time-responsive manner, and further stabilized and preserved rePON1 lactonase activity. Incubation of lyso-DGTS with human serum for 4 h at 37 °C also increased PON1 lactonase activity in a dose-responsive manner. Using tryptophan-fluorescence-quenching assay, lyso-DGTS was found to interact with rePON1 spontaneously with negative free energy (ΔG = -22.87 kJ mol -1 at 25 °C). Thermodynamic parameters and molecular modeling calculations showed that the main interaction of lyso-DGTS with the enzyme is through a hydrogen bond with supporting van der Waals interactions. Furthermore, lyso-DGTS significantly increased rePON1 influx into macrophages and prevented lipid accumulation in macrophages stimulated with oxidized low-density lipid dose-dependently. In vivo supplementation of lyso-DGTS to the circulation of mice fed a high-fat diet via osmotic mini-pumps implanted subcutaneously significantly increased serum PON1 lactonase activity and decreased serum glucose concentrations to the level of mice fed a normal diet. Our findings suggest a beneficial effect of lyso-DGTS on increasing PON1 activity and thus, improving HDL quality and atherosclerotic risk factors. © 2018 BioFactors, 44(3):299-310, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

To study the adverse effects of N-(3-oxododecanoyl)-l-homoserine-lactone (ODHL), a quorum sensing molecule, on mammalian host cells, its effect on membrane potential was examined in rat thymic lymphocytes using flow cytometric techniques with a voltage-sensitive fluorescent probe. As 3-300 μM ODHL elicited hyperpolarization, it is likely that it increases membrane K + permeability because hyperpolarization is directly linked to changing K + gradient across membranes, but not Na + and Cl - gradients. ODHL did not increase intracellular Ca 2+ concentration. ODHL also produced a response in the presence of an intracellular Zn 2+ chelator. Thus, it is unlikely that intracellular Ca 2+ and Zn 2+ are attributed to the response. Quinine, a non-specific K + channel blocker, greatly reduced hyperpolarization. However, because charybdotoxin, tetraethylammonium chloride, 4-aminopyridine, and glibenclamide did not affect it, it is pharmacologically hypothesized that Ca 2+ -activated K + channels, voltage-gated K + channels, and ATP-sensitive K + channels are not involved in ODHL-induced hyperpolarization. Although the K + channels responsible for ODHL-induced hyperpolarization have not been identified, it is suggested that ODHL can elicit hyperpolarization in mammalian host cells, disturbing cellular functions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cellular actions of N-(3-oxododecanoyl)-l-homoserine-lactone (ODHL), a quorum sensing molecule of bacteria, were studied on rat thymocytes using a flow cytometer with appropriate fluorescent dyes to elucidate the effects of ODHL on host cells. A bell-shaped concentration-response relation was observed in the ODHL-induced changes in cellular glutathione content ([GSH]i). ODHL concentration-dependently increased intracellular Zn 2+ levels ([Zn 2+ ]i) and cellular O 2 - content ([O 2 - ]i). The bell-shaped relation induced by ODHL can be explained as follows: a low concentration of ODHL is expected to induce moderate oxidative stress that intracellularly releases Zn 2+ by converting thiols to disulfides. A slight elevation of [Zn 2+ ]i may increase the [GSH]i. On the other hand, it is likely that a high concentration of ODHL causes severe oxidative stress that further causes both the decrease in [GSH]i and the increase in [Zn 2+ ]i. Excessive increase in [Zn 2+ ]i may augment oxidative stress that further decreases the [GSH]i. Other notable actions induced by ODHL included the elevation of [Zn 2+ ]i by Zn 2+ influx and the increase in [GSH]i under Zn 2+ -free conditions. Therefore, it is suggested that ODHL elicits diverse actions on host cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The human mucosal environment in the gut is rich with interactions between microbiota and mammalian epithelia. Microbes such as the Gram-negative bacterium Pseudomonas aeruginosa may use quorum sensing to communicate with other microorganisms and mammalian cells to alter gene expression. Here, we present methodologies to evaluate the effects of P. aeruginosa N-(3-oxo-dodecanoyl)-L-homoserine lactone (3O-C 12 -HSL) on Caco-2 cell monolayers. First, we describe a method for assessing barrier function and permeability of epithelial cells when exposed to 3O-C 12 -HSL by measuring transepithelial electrical resistance (TER) and paracellular flow using fluorescently labeled dextran. Secondly, we detail methods to investigate the effect of 3O-C 12 -HSL on protein-protein interactions of epithelial junction proteins. Lastly, we will detail imaging techniques to visualize Caco-2 barrier disruption following exposure to 3O-C 12 -HSL through the use of confocal laser scanning microscopy (CLSM) and a super resolution technique, stimulated emission depletion (STED) microscopy, to achieve nanoscale visualization of Caco-2 monolayers.

Quorum sensing (QS) is a cell-to-cell communication system that regulates gene expression as a result of the production and perception of signal molecules called autoinducers (AIs). AI-2 is a QS autoinducer produced by both Gram-negative and Gram-positive bacteria, in which it regulates intraspecies and interspecies communication. The identification of QS inhibitors is considered a promising strategy for the development of anti-virulence drugs with reduced selective pressure for resistance. Here we describe a high-throughput virtual screening approach to identify AI-2 quorum sensing inhibitors on the basis of Vibrio harveyi LuxPQ crystal structure. Seven potent inhibitors with IC 50 values in the micromolar range were selected with no effect or low effect on V. harveyi growth rate.

Elevated renal sympathetic nerve activity (RSNA) accompanies a variety of complex disorders, including obstructive sleep apnea, heart failure, and chronic kidney disease. Understanding pathophysiologic renal mechanisms is important for determining why hypertension is both a common sequelae and a predisposing factor of these disorders. The role of the brainstem in regulating RSNA remains incompletely understood. The pedunculopontine tegmentum (PPT) is known for regulating behaviors including alertness, locomotion, and rapid eye movement sleep. Activation of PPT neurons in anesthetized rats was previously found to increase splanchnic sympathetic nerve activity and blood pressure, in addition to altering breathing. The present study is the first investigation of the PPT and its potential role in regulating RSNA. Microinjections of DL-homocysteic acid (DLH) were used to probe the PPT in 100-μm increments in Nembutal-anesthetized rats to identify effective sites, defined as locations where changes in RSNA could be evoked. A total of 239 DLH microinjections were made in 18 rats, which identified 20 effective sites (each confirmed by the ability to evoke a repeatable sympathoexcitatory response). Peak increases in RSNA occurred within 10-20 seconds of PPT activation, with RSNA increasing by 104.5 ± 68.4% (mean ± standard deviation) from baseline. Mean arterial pressure remained significantly elevated for 30 seconds, increasing from 101.6 ± 18.6 mmHg to 135.9 ± 36.4 mmHg. DLH microinjections also increased respiratory rate and minute ventilation. The effective sites were found throughout the rostal-caudal extent of the PPT with most located in the dorsal regions of the nucleus. The majority of PPT locations tested with DLH microinjections did not alter RSNA (179 sites), suggesting that the neurons that confer renal sympathoexcitatory functions comprise a small component of the PPT. The study also underscores the importance of further investigation to determine whether sympathoexcitatory PPT neurons contribute to adverse renal and cardiovascular consequences of diseases such as obstructive sleep apnea and heart failure.

It is well documented that the tumor microenvironment profoundly impacts the etiology and progression of breast cancer, yet the contribution of the resident microbiome within breast tissue remains poorly understood. Tumor microenvironmental conditions, such as hypoxia and dense tumor stroma, predispose progressive phenotypes and therapy resistance, however the role of bacteria in this interplay remains uncharacterized. We hypothesized that the effect of individual bacterial secreted molecules on breast cancer viability and proliferation would be modulated by these tumor-relevant stressors differentially for cells at varying stages of progression. To test this, we incubated human breast adenocarcinoma cells (MDA-MB-231, MCF-DCIS.com) and non-malignant breast epithelial cells (MCF-10A) with N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), a quorum-sensing molecule from Pseudomonas aeruginosa that regulates bacterial stress responses. This molecule was selected because Pseudomonas was recently characterized as a significant fraction of the breast tissue microbiome and OdDHL is documented to impact mammalian cell viability. After OdDHL treatment, we demonstrated the greatest decrease in viability with the more malignant MDA-MB-231 cells and an intermediate MCF-DCIS.com (ductal carcinoma in situ) response. The responses were also culture condition (i.e. microenvironment) dependent. These results contrast the MCF-10A response, which demonstrated no change in viability in any culture condition. We further determined that the observed trends in breast cancer viability were due to modulation of proliferation for both cell types, as well as the induction of necrosis for MDA-MB-231 cells in all conditions. Our results provide evidence that bacterial quorum-sensing molecules interact with the host tissue environment to modulate breast cancer viability and proliferation, and that the effect of OdDHL is dependent on both cell type as well as microenvironment. Understanding the interactions between bacterial signaling molecules and the host tissue environment will allow for future studies that determine the contribution of bacteria to the onset, progression, and therapy response of breast cancer.

Dental plaques are mixed-species biofilms that are related to the development of dental caries. Streptococcus mutans (S. mutans) is an important cariogenic bacterium that forms mixed-species biofilms with Streptococcus gordonii (S. gordonii), an early colonizer of the tooth surface. The LuxS/autoinducer-2(AI-2) quorum sensing system is involved in the regulation of mixed-species biofilms, and AI-2 is proposed as a universal signal for the interaction between bacterial species. In this work, a S. gordonii luxS deficient strain was constructed to investigate the effect of the S. gordonii luxS gene on dual-species biofilm formed by S. mutans and S. gordonii. In addition, AI-2 was synthesized in vitro by incubating recombinant LuxS and Pfs enzymes of S. gordonii together. The effect of AI-2 on S. mutans single-species biofilm formation and cariogenic virulence gene expression were also assessed. The results showed that luxS disruption in S. gordonii altered dual-species biofilm formation, architecture, and composition, as well as the susceptibility to chlorhexidine. And the in vitro synthesized AI-2 had a concentration-dependent effect on S. mutans biofilm formation and virulence gene expression. These findings indicate that LuxS/AI-2 quorum-sensing system of S. gordonii plays a role in regulating the dual-species biofilm formation with S. mutans., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Staphylococcus aureus is an important pathogen that is capable of forming biofilms on biomaterial surfaces to cause biofilm-associated infections. Autoinducer 2 (AI-2), a universal language for interspecies communication, is involved in a variety of physiological activities, although its exact role in Gram-positive bacteria, especially in S. aureus, is not yet thoroughly characterized. Herein we demonstrate that inactivation of luxS, which encodes AI-2 synthase, resulted in increased biofilm formation and higher polysaccharide intercellular adhesion (PIA) production compared with the wild-type strain in S. aureus NCTC8325. The transcript level of rbf, a positive regulator of biofilm formation, was significantly increased in the luxS mutant. All of the parental phenotypes could be restored by genetic complementation and chemically synthesized 4,5-dihydroxy-2,3-pentanedione, the AI-2 precursor molecule, suggesting that AI-2 has a signaling function to regulate rbf transcription and biofilm formation in S. aureus. Phenotypic analysis revealed that the luxS rbf double mutant produced approximately the same amount of biofilms and PIA as the rbf mutant. In addition, real-time quantitative reverse transcription-PCR analysis showed that the icaA transcript level of the rbf mutant was similar to that of the luxS rbf double mutant. These findings demonstrate that the LuxS/AI-2 system regulates PIA-dependent biofilm formation via repression of rbf expression in S. aureus. Furthermore, we demonstrated that Rbf could bind to the sarX and rbf promoters to upregulate their expression., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Quorum sensing regulates a variety of phenotypes in bacteria including the production of virulence factors. Salmonella spp. have quorum sensing systems mediated by three autoinducers (AI-1, AI-2, and AI-3). The AI-1-mediated system is incomplete in that the bacterium relies on the synthesis of signaling molecules by other microorganisms. This study aimed to evaluate the influence of the AI-1 N-dodecanoyl-DL-homoserine lactone (C12-HSL) on the growth, motility, adhesion, and biofilm formation of Salmonella enterica serovar Enteritidis PT4 578 on a polystyrene surface. Experiments were conducted at 37 °C in anaerobic tryptone soy broth supplemented with C12-HSL and/or a mixture of four synthetic furanones, at the concentration of 50 nM each. The planktonic growth, adhesion, swarming, and twitching motility were not altered in the presence of C12-HSL and/or furanones under anaerobic conditions. However, C12-HSL induced biofilm formation after 36 h of cultivation as determined by quantification of biofilm formation, by enumeration of adhered cells to polystyrene coupons, and finally by imaging the presence of multilayered cells on an epifluorescence microscope. When furanones were present in the medium, an antagonistic effect against C12-HSL on the biofilm development was observed. The results demonstrate an induction of biofilm formation in Salmonella Enteritidis by AI-1 under anaerobic conditions. Considering that Salmonella does not produce AI-1 but respond to it, C12-HSL synthesized by other bacterial species could trigger biofilm formation by this pathogen in conditions that are relevant for its pathogenesis.

Objective: Bacterial colonization relies on communication between bacteria via so-called "quorum-sensing molecules", which include the acyl-homoserine lactone group. Certain acyl-homoserine lactones can modulate mammalian cell function and are thought to contribute to bacterial pathogenicity. Given the role of mast cells in host defense, we investigated the ability of acyl-homoserine lactones to modulate mast cell function., Methods: We utilized murine primary mast cell cultures to assess the effect of acyl-homoserine lactones on degranulation and cytokine release in response to different stimuli. We also assessed cell migration in response to chemoattractants. The effect of acyl-homoserine lactones in vivo was tested using a passive cutaneous anaphylaxis model., Results: Two of the tested quorum-sensing molecules, N-3-oxo-dodecanoyl-L-homoserine lactone and N-Dodecanoyl-L-homoserine lactone, inhibited IgE dependent and independent degranulation and mediator release from primary mast cells. Further testing of N-3-oxo-dodecanoyl-L-homoserine lactone, the most potent inhibitor and a product of Pseudomonas aeruginosa, revealed that it also attenuated chemotaxis and LPS induced cytokine production. In vivo, N-3-oxo-dodecanoyl-L-homoserine lactone inhibited the passive cutaneous anaphylaxis response in mice., Conclusion: The ability of N-3-oxo-dodecanoyl-L-homoserine lactone to stabilize mast cells may contribute to the pathogenicity of P. aeruginosa but could potentially be exploited therapeutically in allergic disease.

Highly fluorinated amino acids can stabilize proteins and complexes with proteins, via enhanced hydrophobicity, and provide novel methods for identification of specific molecular events in complex solutions, via selective detection by 19 F NMR and the absence of native 19 F signals in biological contexts. However, the potential applications of 19 F NMR in probing biological processes are limited both by the strong propensities of most highly fluorinated amino acids for the extended conformation and by the relatively modest sensitivity of NMR spectroscopy, which typically constrains measurements to mid-micromolar concentrations. Herein, we demonstrate that perfluoro-tert-butyl homoserine exhibits a propensity for compact conformations, including α-helix and polyproline helix (PPII), that is similar to that of methionine. Perfluoro-tert-butyl homoserine has nine equivalent fluorines that do not couple to any other nuclei, resulting in a sharp singlet that can be sensitively detected rapidly at low micromolar concentrations. Perfluoro-tert-butyl homoserine was incorporated at sites of leucine residues within the α-helical LXXLL short linear motif of estrogen receptor (ER) coactivator peptides. A peptide containing perfluoro-tert-butyl homoserine at position i + 3 of the ER coactivator LXXLL motif exhibited a K d of 2.2 μM for the estradiol-bound estrogen receptor, similar to that of the native ligand. 19 F NMR spectroscopy demonstrated the sensitive detection (5 μM concentration, 128 scans) of binding of the peptide to the ER and of inhibition of protein-protein interaction by the native ligand or by the ER antagonist tamoxifen. These results suggest diverse potential applications of perfluoro-tert-butyl homoserine in probing protein function and protein-protein interfaces in complex solutions.

Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti- S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis .

A Pseudomonas aeruginosa quorum-sensing system, which produces N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C 12 -HSL) and N-butanoyl-l-homoserine lactone (C 4 -HSL), regulates the virulence factors. In our previous study, 3-oxo-C 12 -HSL, encoded by lasI gene, was shown to promote wound healing. However, the effect of C 4 -HSL, encoded by rhlI gene, remains to be elucidated. We addressed the effect of C 4 -HSL on wounds in P. aeruginosa infection. Wounds were created on the backs of Sprague-Dawley SD rats, and P. aeruginosa PAO1 (PAO1) or its rhlI deletion mutant (ΔrhlI) or lasI deletion mutant (ΔlasI) was inoculated onto the wound. Rats were injected intraperitoneally with anti-C 4 -HSL antiserum or treated with C 4 -HSL at the wound surface. PAO1 inoculation led to significant acceleration of wound healing, which was associated with neutrophil infiltration and TNF-α synthesis. These responses were reversed, except for TNF-α production, when ΔrhlI was inoculated instead of PAO1 or when rats were co-treated with PAO1 and anti-C 4 -HSL antiserum. In contrast, the healing process and neutrophil infiltration, but not TNF-α synthesis, were accelerated when C 4 -HSL was administered in the absence of PAO1. This acceleration was not affected by anti-TNF-α antibody. These results suggest that C 4 -HSL may be involved in the acceleration of acute wound healing in P. aeruginosa infection by modifying the neutrophilic inflammation., (© 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

Bacterial biofilms are a common cause of chronic medical implant infections. Treatment and eradication of biofilms by conventional antibiotic therapy has major drawbacks including toxicity and side effects associated with high-dosage antibiotics. Additionally, administration of high doses of antibiotics may facilitate the emergence of antibiotic resistant bacteria. Thus, there is an urgent need for the development of treatments that are not based on conventional antibiotic therapies. Presented herein is a novel bacterial biofilm combination treatment independent of traditional antibiotics, by using low electric fields in combination with small molecule inhibitors of bacterial quorum sensing - autoinducer-2 analogs. We investigate the effect of this treatment on mature Escherichia coli biofilms by application of an alternating and offset electric potential in combination with the small molecule inhibitor for 24 h using both macro and micro-scale devices. Crystal violet staining of the macro-scale biofilms shows a 46 % decrease in biomass compared to the untreated control. We demonstrate enhanced treatment efficacy of the combination therapy using a high-throughput polydimethylsiloxane-based microfluidic biofilm analysis platform. This microfluidic flow cell is designed to reduce the growth variance of in vitro biofilms while providing an integrated control, and thus allows for a more reliable comparison and evaluation of new biofilm treatments on a single device. We utilize linear array charge-coupled devices to perform real-time tracking of biomass by monitoring changes in optical density. End-point confocal microscopy measurements of biofilms treated with the autoinducer analog and electric fields in the microfluidic device show a 78 % decrease in average biofilm thickness in comparison to the negative controls and demonstrate good correlation with real-time optical density measurements. Additionally, the combination treatment showed 76 % better treatment efficacy compared to conventional antibiotic therapy. Taken together these results suggest that the antibiotic-free combination treatment described here may provide an effective alternative to traditional antibiotic therapies against bacterial biofilm infections. Use of this combination treatment in the medical and environmental fields would alleviate side effects associated with high-dosage antibiotic therapies, and reduce the rise of antibiotic-resistant bacteria.

A mucus layer coats the gastrointestinal tract and serves as the first line of intestinal defense against infection. N-acyl-homoserine lactone (AHL) quorum-sensing molecules produced by gram-negative bacteria in the gut can influence the homeostasis of intestinal epithelium. In this study, we investigated the effects of two representative long- and short-chain AHLs, N-3-(oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl homoserine lactone (C4-HSL), on cell viability and mucus secretion in LS174T cells. C12-HSL but not C4-HSL significantly decreased cell viability by inducing mitochondrial dysfunction and activating cell apoptosis which led to a decrease in mucin expression. Pretreatment with lipid raft disruptor (Methyl-β-cyclodextrin, MβCD) and oxidative stress inhibitor (N-acetyl-L-cysteine, NAC) slightly rescued the viability of cells damaged by C12-HSL exposure, while the paraoxonase 2 (PON2) inhibitor (Triazolo[4,3-a]quinolone, TQ416) significantly affected recovering cells viability and mucin secretion. When LS174T cells were treated with C12-HSL and TQ416 simultaneously, TQ416 showed the maximal positive effect on cells viability. However, if cells were first treated with C12-HSL for 40 mins, and then TQ46 was added, the TQ416 had no effect on cell viability. These results suggest that the C12-HSL-acid process acts at an early step to activate apoptosis as part of C12-HSL's effect on intestinal mucus barrier function.

Quorum sensing (QS) communication allows Pseudomonas aeruginosa to collectively control its population density and the production of biofilms and virulence factors. QS signal molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C12-HSL), can also affect the behavior of host cells, e.g., by modulating the chemotaxis, migration, and phagocytosis of human leukocytes. Moreover, host water homeostasis and water channels aquaporins (AQP) are critical for cell morphology and functions as AQP interact indirectly with the cell cytoskeleton and signaling cascades. Here, we investigated how P. aeruginosa 3O-C12-HSL affects cell morphology, area, volume and AQP9 expression and distribution in human primary macrophages, using quantitative PCR, immunoblotting, two- and three-dimensional live imaging, confocal and nanoscale imaging. Thus, 3O-C12-HSL enhanced cell volume and area and induced cell shape and protrusion fluctuations in macrophages, processes tentatively driven by fluxes of water across cell membrane through AQP9, the predominant AQP in macrophages. Moreover, 3O-C12-HSL upregulated the expression of AQP9 at both the protein and mRNA levels. This was accompanied with enhanced whole cell AQP9 fluorescent intensity and redistribution of AQP9 to the leading and trailing regions, in parallel with increased cell area in the macrophages. Finally, nanoscopy imaging provided details on AQP9 dynamics and architecture within the lamellipodial area of 3O-C12-HSL-stimulated cells. We suggest that these novel events in the interaction between P. aeruginosa and macrophage may have an impact on the effectiveness of innate immune cells to fight bacteria, and thereby resolve the early stages of infections and inflammations.

Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells.