Identification of boronic acids as antagonists of bacterial quorum sensing in Vibrio harveyi

Bacterial quorum sensing plays a very important role in the regulation of biofilm formation, virulence, conjugation, sporulation, and swarming mobility. Inhibitors of bacterial quorum sensing are important research tools and potential therapeutic agents. In this paper, we describe for the first time the discovery of several boronic acids as single digit micromolar inhibitors of bacterial quorum sensing in Vibrio harveyi.     

Dihydropyrrolones as bacterial quorum sensing inhibitors

Bacteria regulate their pathogenicity and biofilm formation through quorum sensing (QS), which is an intercellular communication system mediated by the binding of signaling molecules to QS receptors such as LasR. In this study, a range of dihydropyrrolone (DHP) analogues were synthesized via the lactone-lactam conversion of lactone intermediates. The synthesized compounds were tested for their ability to inhibit QS, biofilm formation and bacterial growth of Pseudomonas aeruginosa. The compounds were also docked into a LasR crystal structure to rationalize the observed structure-activity relationships. The most active compound identified in this study was compound 9i, which showed 63.1% QS inhibition of at 31.25?µM and 60% biofilm reduction at 250?µM with only moderate toxicity towards bacterial cell growth.     

Bacterial interspecies quorum sensing in the mammalian gut microbiota

The mammalian gastrointestinal tract harbors a diverse and complex resident bacterial community, which interacts with the host in many beneficial processes required for optimal host health. We are studying the importance of bacterial cell-cell communication mediated by the interspecies quorum-sensing signal autoinducer-2 (AI-2) in the beneficial properties of the gut microbiota. Our recent work provided the first evidence that AI-2 produced by Escherichia coli can influence the species composition of this community in the mouse gut. We showed that, under conditions of microbiota imbalances induced by antibiotic treatments, E. coli, which increases intestinal AI-2 levels, not only had an effect on the overall structure of the microbiota community, but specifically favored the expansion of the Firmicutes phylum. Because the Firmicutes are very important for many gut functions and were the group of bacteria most severely affected by antibiotic treatment with streptomycin, we are addressing the possibility that AI-2 can influence the balance of the major bacterial groups in the gut and promote recovery of gut homeostasis. Overall, we want to understand how bacterial chemical signaling shapes the multi-species bacterial communities in the mammalian gut and how these communities affect host physiology.     

Vibrio harveyi hemolysin induces ultrastructural changes and apoptosis in flounder (Paralichthys olivaceus) cells

Vibrio harveyi hemolysin (VHH) is considered a major pathogenic virulence factor to fish. However, the VHH active-site mutant has lost all hemolytic and phospholipase activities as well as pathogenicity. In this study, the effect of VHH on erythrocytes and a gill cell line from flounder was elucidated. Erythrocyte membranes formed thin tubular protrusions immediately after exposure to VHH, and membrane corrugations were evident after extended incubation. In contrast, the mutant VHH did not induce any gross morphological changes. With VHH-treated FG-9307 cells, a cell line derived from flounder gill, destruction of organelles and formation of features resembling apoptotic bodies were observed. Immunogold staining showed that a large amount of VHH was deposited on the membranes and membrane debris of erythrocytes and FG-9307 cells after treatment with VHH. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in VHH-treated FG-9307 cells using DAPI staining. Moreover, cell cycle analysis showed that VHH increased the proportion of cells in G1 phase. In addition, VHH significantly increased the percentage of apoptosis, the number of TUNEL positive apoptotic cells, and caspase-3 activity in FG-9307 cells when compared with the untreated controls. These data suggested that VHH killed the cells through apoptosis via the caspase activation pathway.     

Extracts of seaweeds as potential inhibitors of quorum sensing and bacterial growth

Macroalgae are an important source of antimicrobial compounds. However, it is unclear if these compounds are produced by the algae themselves, by their associated bacteria, or by both. The main aim of this study was to investigate the potential of macroalgae and their associated microorganisms to inhibit bacterial quorum sensing (QS) and growth. Before extraction, half of the algal specimens were treated with 30% ethanol to remove surface associated bacteria. Canistrocarpus cervicornis extracts were able to inhibit QS of the reporter Chromobacterium violaceum CV017, where extracts with associated bacteria were more efficient than those without bacteria. However, not all algal extracts that inhibited QS of CV017 were able to inhibit bacterial attachment of Pseudomonas aeruginosas PA01, showing specific activity of algal metabolites. Only 58% of the extracts showed antibacterial activity against eight marine fouling and pathogenic bacterial strains tested. Our data suggests that algae and their associated microbiota are important sources of antimicrobial compounds which potentially can be used in future biotechnological applications.     

A simple assay of cyclic-AMP phosphodiesterase in the presence of interfering enzymes in Vibrio harveyi

PEI-cellulose thin layer chromatography is used to separate the reaction products formed from cyclic-AMP initiated by phosphodiesterase in crude extracts of bacteria.     

Molecular mechanisms of bacterial quorum sensing as a new drug target

Bacterial quorum sensing evolved as a means for bacterial communities to rapidly and coordinately change genome expression patterns in response to environmental cues. Each cell in a community produces and responds to a signaling molecule called an autoinducer that serves as an indicator of the population density. A high concentration of autoinducer is associated with a large, often confined population, which requires altered gene expression for survival. Quorum sensing is one mechanism through which a bacterial population receives input from the environment and elicits an appropriate response. Because many pathogens require quorum sensing to produce virulence factors in response to association with a human host, the signaling pathway is a target for design of small-molecule inhibitors.     

Identification of natural product compounds as quorum sensing inhibitors in Pseudomonas fluorescens P07 through virtual screening

Pseudomonas fluorescens, a Gram-negative psychrotrophic bacteria, is the main microorganism causing spoilage of chilled raw milk and aquatic products. Quorum sensing (QS) widely exists in bacteria to monitor their population densities and regulate numerous physiological activities, such as the secretion of siderophores, swarming motility and biofilm formation. Thus, searching for quorum sensing inhibitors (QSIs) may be another promising way to control the deterioration of food caused by P. fluorescens. Here, we screened a traditional Chinese medicine (TCM) database to discover potential QSIs with lesser toxicity. The gene sequences of LuxI- and LuxR-type proteins of P. fluorescens P07 were obtained through whole-genome sequencing. In addition, the protein structures built by homology modelling were used as targets to screen for QSIs. Twenty-one compounds with a dock score greater than 6 were purchased and tested by biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136). The results showed that 10 of the compounds were determined as hits (hit rate: 66.67%). Benzyl alcohol, rhodinyl formate and houttuynine were effective QSIs. The impact of the most active compound (benzyl alcohol) on the phenotypes of P. fluorescens P07, including swimming and swarming motility, production of extracellular enzymes and siderophores, N-acylhomoserine lactone (AHLs) content and biofilm formation were determined. The inhibitory mechanism of benzyl alcohol on the QS system of P. fluorescens P07 is further discussed. This study reveals the feasibility of searching for novel QSIs through virtual screening.     

Evidence for quorum sensing in Clostridium botulinum 56A

AIMS: Experiments were designed to detect quorum-sensing signals produced by Clostridium botulinum. METHODS AND RESULTS: Clostridium botulinum 56A cell-free supernatants obtained at the end of lag phase, the mid-exponential phase and early stationary phase of growth were assayed for bioluminescence in the Vibrio harveyi quorum-sensing assay system. Twelve and 16-h culture supernatants induced bioluminescence in the auto-inducer 2 (AI-2) but not the auto-inducer 1 (AI-1) assay. Intra-species quorum sensing was also assayed as the ability of the supernatants to promote spore germination and outgrowth in a microtitre plate system. Spore populations exposed to C. botulinum supernatant from the end of lag phase became positive for growth sooner than controls. CONCLUSIONS: The influence of cell-free supernatant on ungerminated spores and detection of bioluminescence in the AI-2 assay are evidence for a signalling molecule(s) and provide a first step in characterizing C. botulinum quorum sensing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that spores do not behave independently of each other and may explain the inocula size effects observed in challenge studies. Whether AI-2 production in C. botulinum serves as an inter-species signal or as a detoxification mechanism remains to be determined.     

群体感应抑制剂对海洋生态功能菌生物膜形成的影响

[目的]研究天然群体感应抑制剂(Quorum sensing inhibitors,QSI)分子对海洋生态功能菌生物膜形成的影响.[方法]以对污损生物幼虫附着具有诱导作用的海洋细菌为目标菌,通过在其生物膜的形成过程中添加天然群体感应抑制剂,研究其对目标菌成膜细菌数和浮游细菌数、生物膜形态以及生物膜表面胞外多糖含量的影响.[结果]呋喃酮和吡啶在50 mg/L时,对8株目标菌的成膜有显著的抑制作用,抑制率在80％左右,吲哚、青霉烷酸和香豆素在较高浓度800 mg/L才有比较好的抑制活性.生长抑制实验结果显示,同等浓度下,QSI分子对目标菌成膜的抑制活性明显高于其对浮游细菌生长的抑制活性.结果表明,QSI分子主要通过干扰目标菌群体感应系统以抑制生物膜的形成.[结论]研究证实QSI分子在海洋菌生物膜形成过程中具有一定的调控作用.通过添加QSI可能能够间接抑制由生物膜诱导的污损生物附着,从而以新的角度研制新型抗污损物质.     

具有细菌群体感应抑制活性海洋来源真菌的筛选鉴定

许多致病菌的致病机制依赖于群体感应系统的调控,经实验证明群体感应系统突变或缺失的菌株致病能力显著下降,筛选高效的群体感应抑制剂有望成为解决细菌感染以及细菌耐药性问题的一个有效途径。从海洋软体动物体内分离海洋真菌69株,发酵液粗提物经QSIS2 (Quorum Sensing Inhibitor Selector 2) 筛选模型和紫色杆菌CV026指示菌株筛选后得到编号QY013的粗提物具有群体感应抑制活性,进一步实验表明该粗提物能够显著降低铜绿假单胞菌群体感应调控的毒力因子绿脓菌素的产量,以及紫色杆菌群体感应调控的紫色菌素的产量,且在有效浓度范围内对细菌生长不产生影响。形态学特征和18S rDNA序列分析表明菌株QY013为Penicillium属。文中筛选到一株具有细菌群体感应抑制活性的海洋来源真菌,其发酵液粗提物中的有效活性成分可用于新型抗菌药物的研究。     

A mathematical model of quorum sensing in a growing bacterial biofilm

In a process called quorum sensing, bacteria monitor their population density via extracellular signaling molecules and modulate gene expression accordingly. This paper describes a one-dimensional model of a growing Pseudomonas aeruginosa biofilm. Quorum sensing has been included in the model by the addition of equations describing the production, degradation, and diffusion of acyl-homoserine lactones in the biofilm. In order for quorum sensing to initiate near the substratum, in accordance with experimental observations, model results suggest that cells in oxygen-deficient regions of the biofilm must still be synthesizing the signal compound. This result highlights the importance of careful study of the relationship between metabolic activity of the bacterium and signal synthesis. Received 11 March 2002/ Accepted in revised form 01 August 2002     

A mathematical model of the Gac/Rsm quorum sensing network in Pseudomonas fluorescens

I present a deterministic model of the dynamics of signal transduction and gene expression in the Gac/Rsm network of the soil-dwelling bacterium Pseudomonas fluorescens. The network is involved in quorum sensing and governs antifungal production in this important biocontrol agent. A central role is played by small untranslated RNAs, which sequester regulatory mRNA-binding proteins. The model provides a reasonable match to the available data, which consists primarily of time series from reporter gene fusions. I use the model to investigate the information-processing properties of the Gac/Rsm network, in part by comparing it to a simplified model capable of quorum sensing. The results suggest that the complexity and redundancy of the Gac/Rsm network have evolved to meet the conflicting requirements of high sensitivity to environmental conditions and a conservative, robust response to variability in parameter values. Similar systems exist in a wide variety of bacteria, where they control a diverse set of population-dependent behaviors. This makes them important subjects for mathematical models that can help link empirical understanding of network structure to theoretical insights into how these networks have evolved to function under natural conditions.     

Identification by Inverse Virtual Screening of magnolol-based scaffold as new tankyrase-2 inhibitors

The natural product magnolol (1) and a selection of its bioinspired derivatives 2–5, were investigated by Inverse Virtual Screening in order to identify putative biological targets from a panel of 308 proteins involved in cancer processes. By this in silico analysis we selected tankyrase-2 (TNKS2), casein kinase 2 (CK2) and bromodomain 9 (Brd9) as potential targets for experimental evaluations. The Surface Plasmon Resonance assay revealed that 3–5 present a good affinity for tankyrase-2, and, in particular, 3 showed an antiproliferative activity on A549 cells higher than the well-known tankyrase-2 inhibitor XAV939 used as reference compound.     

Bicyclic brominated furanones: A new class of quorum sensing modulators that inhibit bacterial biofilm formation

Both natural and synthetic brominated furanones are known to inhibit biofilm formation by bacteria, but their toxicity to mammalian cells is often not reported. Here, we designed and synthesized a new class of brominated furanones (BBFs) that contained a bicyclic structure having one bromide group with well-defined regiochemistry. This class of molecules exhibited reduction in the toxicity to mammalian cells (human neuroblastoma SK-N-SH) and did not inhibit bacteria (Pseudomonas aeruginosa and Escherichia coli) growth, but retained the inhibitory activity towards biofilm formation of bacteria. In addition, all the BBFs inhibited the production of virulence factor elastase B in P. aeruginosa. To explore the effect of BBFs on quorum sensing, we used a reporter gene assay and found that 6-BBF and 7-BBF exhibited antagonistic activities for LasR protein in the lasI quorum sensing circuit, while 5-BBF showed agonistic activity for the rhlI quorum sensing circuit. This study suggests that structural variation of brominated furanones can be designed for targeted functions to control biofilm formation.     

Effects of quorum sensing on cell viability in Streptococcus mutans biofilm formation

Streptococcus mutans (S. mutans) uses a quorum sensing (QS) signaling system, which is dependent on competence stimulating peptide (CSP), to regulate diverse physiological activities including bacteriocin production, genetic transformation, and biofilm formation. However, the mechanism of the QS system-induced biofilm formation remains unclear. Here, we demonstrated that the late-stage biofilm formation was increased by the addition of exogenous CSP in S. mutans. The numbers of dead cells in biofilms formed in presence of CSP was 64.5% higher than that without CSP after 12 h (p < 0.05) and 76.3% higher after 24 h (p < 0.05), the numbers of live cells in biofilms formed in presence of CSP were 89.3% higher than that without CSP after 24 h (p < 0.01). The expression of QS-associated genes was increased 3.4-5.3-fold by CSP in biofilms. Our results revealed that cell viability of S. mutans grown in biofilms is affected by the CSP-dependent QS system.     

Detection of quorum sensing signal molecules in the family Vibrionaceae

Aims: The aim of this study was to detect the production of three kinds of quorum sensing (QS) signal molecules, i.e. the N‐acyl‐homoserine lactone (AHL), the autoinducer‐2 (AI‐2) and the cholerae autoinducer‐1‐like (CAI‐1‐like) molecules in 25 Vibrionaceae strains. Methods and Results: The QS signal molecules in 25 Vibrionaceae strains were detected with different biosensors. Except Salinivibrio costicola VIB288 and Vibrio natriegens VIB299, all the other 23 Vibrionaceae strains could produce one or more kinds of detectable QS signal molecules. Twenty‐one of the 25 strains were found to produce AHL signal molecules by using Vibrio harveyi JMH612 and Agrobacterium tumefaciens KYC55 (pJZ372; pJZ384; pJZ410) as biosensors. The AHL fingerprints of eight strains were detected by thin‐layer chromatography with Ag. tumefaciens KYC55, and two of them, i.e. V. mediterranei VIB296 and Aliivibrio logei VIB414 had a high diversity of AHLs. Twenty of the 25 strains were found to have the AI‐2 activity, and the luxS gene sequences in 18 strains were proved to be conserved by PCR amplification and sequencing. Only six (five Vibrio strains and A. logei VIB414) of the 25 strains possessed the CAI‐1‐like activity. A. logei VIB414, V. campbellii VIB285, V. furnissii VIB293, V. pomeroyi LMG20537 and two V. harveyi strains VIB571 and VIB645 were found to produce all the three kinds of QS signal molecules. Conclusions: The results indicated that the QS signal molecules, especially AHL and AI‐2 molecules, were widespread in the family Vibrionaceae. Significance and Impact of the Study: In response to a variety of environmental conditions and selection forces, the family Vibrionaceae produced QS signal molecules with great diversity and complexity. The knowledge we obtained from this study will be useful for further research on the roles of different QS signal molecules in this family.     

Synthetic nanoparticles for selective hydrolysis of bacterial autoinducers in quorum sensing

N-acyl homoserine lactones (AHLs) are signal molecules used by a large number of gram-negative bacteria in quorum sensing and their hydrolysis is known to inhibit biofilm formation. Micellar imprinting of AHL-like templates with catalytic functional monomers yielded water-soluble nanoparticles with AHL-shaped active site and nearby catalytic groups. Either Lewis acidic zinc ions or nucleophilic pyridyl ligands could be introduced through this strategy, yielding artificial enzymes for the hydrolysis of AHLs in a substrate-selective fashion.     

Inactivation of bacterial quorum sensing signals N-acyl homoserine lactones is widespread in yeasts

The inactivation of quorum sensing signals, a phenomenon known as quorum quenching, has been described in diverse microorganisms, though it remains almost unexplored in yeasts. Beyond the well-known properties of these microorganisms for the industry or as eukaryotic models, the role of yeasts in soil or in the inner tissues of a plant is largely unknown. In this report, the wider survey of quorum quenching activities in yeasts isolated from Antarctic soil and the inner tissues of sugarcane, a tropical crop, is presented. Results show that, independently of their niche, quorum quenching activities are broadly present in unicellular fungi. Although yeasts showing a broad range of quorum quenching activity are present in the two niches, at the same time specific AHL inactivation profiles can also be found. Furthermore, yeasts from both sampling sites show quorum quenching activities compatible with lactonase-like and acylase-like inactivations of AHLs. Interestingly, the characterization of Rhodotorula mucilaginosa 7Apo1 showed that the presence of a particular AHL does not interfere with the quenching of a second molecule. Evidence suggests that yeasts could play a role in the modulation of the quorum sensing activity of bacteria. The relationship among phylogeny, sampling sites and yeast quorum quenching activities of the isolates is analyzed.