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.     

Monitoring of Vibrio harveyi quorum sensing activity in real time during infection of brine shrimp larvae

Quorum sensing, bacterial cell-to-cell communication, has been linked to the virulence of pathogenic bacteria. Indeed, in vitro experiments have shown that many bacterial pathogens regulate the expression of virulence genes by this cell-to-cell communication process. Moreover, signal molecules have been detected in samples retrieved from infected hosts and quorum sensing disruption has been reported to result in reduced virulence in different host–pathogen systems. However, data on in vivo quorum sensing activity of pathogens during infection of a host are currently lacking. We previously reported that quorum sensing regulates the virulence of Vibrio harveyi in a standardised model system with gnotobiotic brine shrimp (Artemia franciscana) larvae. Here, we monitored quorum sensing activity in Vibrio harveyi during infection of the shrimp, using bioluminescence as a read-out. We found that wild-type Vibrio harveyi shows a strong increase in quorum sensing activity early during infection. In this respect, the bacteria behave remarkably similar in different larvae, despite the fact that only half of them survive the infection. Interestingly, when expressed per bacterial cell, Vibrio harveyi showed around 200-fold higher maximal quorum sensing-regulated bioluminescence when associated with larvae than in the culture water. Finally, the in vivo quorum sensing activity of mutants defective in the production of one of the three signal molecules is consistent with their virulence, with no detectable in vivo quorum sensing activity in AI-2- and CAI-1-deficient mutants. These results indicate that AI-2 and CAI-1 are the dominant signals during infection of brine shrimp.     

Bacterial quorum sensing in symbiotic and pathogenic relationships with hosts*

Gram-negative bacteria communicate with each other by producing and sensing diffusible signaling molecules. This mechanism is called quorum sensing (QS) and regulates many bacterial activities from gene expression to symbiotic/pathogenic interactions with hosts. Therefore, the elucidation and control of bacterial QS systems have been attracted increasing attention over the past two decades. The most common QS signals in Gram-negative bacteria are N-acyl homoserine lactones (AHLs). There are also bacteria that employ different QS systems, for example, the plant pathogen Ralstonia solanacearum utilizes 3-hydroxy fatty acid methyl esters as its QS signals. The QS system found in the endosymbiotic bacterium associated with the fungus Mortierella alpina, the development of an affinity pull-down method for AHL synthases, and the elucidation of a unique QS circuit in R. solanacearum are discussed herein.     

Inhibition of Pseudomonas aeruginosa quorum sensing by AI-2 analogs

Autoinducer-2 (AI-2) has been suggested to serve as a universal interspecies quorum sensing signaling molecule. We have synthesized a set of AI-2 analogs with small incremental changes in alkyl substitution on C-2 and evaluated them for their agonistic and antagonistic potential as quorum sensing (QS) attenuators in two different bacterial species: Pseudomonas aeruginosa and Vibrio harveyi. Unexpectedly, several of the analogs were found to function as synergistic QS agonists in V. harveyi, while two of these analogs inhibit QS in P. aeruginosa.     

One step preparation and electrochemical analysis of IQS,a cell–cell communication signal in the nosocomial pathogen Pseudomonas aeruginosa

Pseudomonas aeruginosa uses a hierarchical cell–cell communication system consisting of a number of regulatory elements to coordinate the expression of bacterial virulence genes. Sensitive detection of quorum sensing (QS) molecules has the potential for early identification of P. aeruginosa facilitating early medical intervention. A recently isolated cell–cell communication molecule, a thiazole termed IQS, can bypass the las QS system of P. aeruginosa under times of stress, activating a subset of QS-controlled genes. This compound offers a new target for pathogen detection and has been prepared in a one step protocol. A simple electrochemical strategy was employed for its sensitive detection using boron-doped diamond and glassy carbon electrodes by cyclic voltammetry and amperometry.     

Silencing the mob: disrupting quorum sensing as a means to fight plant disease

Bacteria are able to sense their population's density through a cell–cell communication system, termed ‘quorum sensing’ (QS). This system regulates gene expression in response to cell density through the constant production and detection of signalling molecules. These molecules commonly act as auto‐inducers through the up‐regulation of their own synthesis. Many pathogenic bacteria, including those of plants, rely on this communication system for infection of their hosts. The finding that the countering of QS‐disrupting mechanisms exists in many prokaryotic and eukaryotic organisms offers a promising novel method to fight disease. During the last decade, several approaches have been proposed to disrupt QS pathways of phytopathogens, and hence to reduce their virulence. Such studies have had varied success in vivo, but most lend promising support to the idea that QS manipulation could be a potentially effective method to reduce bacterial‐mediated plant disease. This review discusses the various QS‐disrupting mechanisms found in both bacteria and plants, as well as the different approaches applied artificially to interfere with QS pathways and thus protect plant health.     

Bacterial quorum sensing: circuits and applications

Bacterial quorum sensing (QS) systems are cell density—dependent regulatory networks that coordinate bacterial behavioural changes from single cellular organisms at low cell densities to multicellular types when their population density reaches a threshold level. At this stage, bacteria produce and perceive small diffusible signal molecules, termed autoinducers in order to mediate gene expression. This often results in phenotypic shifts, like planktonic to biofilm or non-virulent to virulent. In this way, they regulate varied physiological processes by adjusting gene expression in concert with their population size. In this review we give a synopsis of QS mediated cell–cell communication in bacteria. The first part focuses on QS circuits of some Gram-negative and Gram-positive bacteria. Thereafter, attention is drawn on the recent applications of QS in development of synthetic biology modules, for studying the principles of pattern formation, engineering bi-directional communication system and building artificial communication networks. Further, the role of QS in solving the problem of biofouling is also discussed.     

细菌中群体感应调节系统

细菌根据特定信号分子的浓度可以监测周围环境中自身或其它细菌的数量变化,当信号达到一定的浓度阈值时,能启动菌体中相关基因的表达来适应环境中的变化,这一调控系统被称为细菌的群体感应调节系统(QuorumSensing系统)。本文系统介绍了细菌感知种内与种间数量的群体感应调节系统,并阐述了植物针对病原菌这一信号系统的抗病策略。     

Robust and sensitive control of a quorum‐sensing circuit by two interlocked feedback loops

The quorum‐sensing (QS) response of Vibrio fischeri involves a rapid switch between low and high induction states of the lux operon over a narrow concentration range of the autoinducer (AI) 3‐oxo‐hexanoyl‐L ‐homoserine lactone. In this system, LuxR is an AI‐dependent positive regulator of the lux operon, which encodes the AI synthase. This creates a positive feedback loop common in many bacterial species that exhibit QS‐controlled gene expression. Applying a combination of modeling and experimental analyses, we provide evidence for a LuxR autoregulatory feedback loop that allows LuxR to increase its concentration in the cell during the switch to full lux activation. Using synthetic lux gene fragments, with or without the AI synthase gene, we show that the buildup of LuxR provides more sensitivity to increasing AI, and promotes the induction process. Elevated LuxR levels buffer against spurious variations in AI levels ensuring a robust response that endows the system with enhanced hysteresis. LuxR autoregulation also allows for two distinct responses within the same cell population.     

The Distinct Quorum Sensing Hierarchy of las and rhl in Pseudomonas sp. M18

Pseudomonas sp. M18 is a rhizosphere isolate capable of producing two kinds of antifungal agents: phenazine-1-carboxylic acid (PCA) and pyoluteorin. Recently, the two well-studied quorum sensing (QS) systems of Pseudomonas aeruginosa, LasR/LasI and RhlR/RhlI, have also been identified in this strain. However, in this study, through the use of lacZ translational fusion expression analysis and acyl-homoserine lactone thin-layer chromatography (TLC) bioassays, we clearly display a more complex and distinctive hierarchy of the las and rhl QS systems in strain M18. In this QS cascade, expression of rhlI was negatively controlled by the LasR/LasI QS system. In contrast with lasI, which negatively regulated the rhlR induction, lasR exerted a positive influence on rhlR expression during the log-phase. This interrelationship indicated that the response regulators (LasR and RhlR) of the QS system are expressed independently of their cognate synthases (LasI and RhlI). Furthermore, the las system also modulated the timing and magnitude of the rhlI and rhlR maximal expression. In addition, our data imply that the lasR gene exerts its negative control on PCA production through modulation of rhlI expression. Thus, interactions between the two QS systems are strain specific.     

Effects of temperature,growth phase and luxO-disruption on regulation systems of toxin production in Vibrio vulnificus strain L-180, a human clinical isolate

Vibrio vulnificus is a halophilic estuarine bacterium while it causes fatal septicemia or necrotizing wound infections in humans. This pathogen secretes the metalloprotease (V. vulnificus protease: VVP) and the cytolysin (V. vulnificus hemolysin: VVH) as protein toxins; however, their production was coordinated in response to the bacterial cell density. This regulation is termed quorum sensing (QS) and is mediated by the small diffusible molecule called autoinducer 2 (AI-2). In the present study, we investigated effects of disruption of luxO encoding a central response regulator of the QS circuit, as well as effects of temperature and growth phase, on the toxin production by V. vulnificus. Disruption of luxO was found to increase VVP production and expression of its gene vvpE. The expression of smcR, crp and rpoS, of which products positively regulate vvpE expression, and luxS encoding the AI-2 synthetase were also significantly increased. On the other hand, the luxO disruption resulted in reduction of VVH production and expression of its gene vvhA. Expression of other two genes affecting the QS circuit, luxT and rpoN, were also significantly decreased. The regulation systems of VVP production were found to exert their action during the stationary phase of the bacterial growth and to be operated strongly at 26 °C. By contrast, those of VVH production apparently started at the log phase and were operated more effectively at 37 °C.     

Vibrio vulnificus quorum-sensing system operates in cirrhotic ascites, a human ex vivo experimental system

This study was conducted to experimentally determine whether a Vibrio vulnificus quorum sensing (QS) system actually operates ex vivo. The expressions of luxS encoding autoinducer-2 synthase, of smcR encoding the master regulator of QS system, and of vvpE encoding a metalloprotease, the best known target directly regulated by QS, were increased with increasing bacterial density in cirrhotic ascites used as a human ex vivo experimental system. A luxS or smcR mutation significantly decreased vvpE expression under the same conditions. Accordingly, V. vulnificus QSS is likely to operate ex vivo.     

Furvina inhibits the 3-oxo-C12-HSL-based quorum sensing system of Pseudomonas aeruginosa and QS-dependent phenotypes

Disruption of cell–cell communication or quorum sensing (QS) is considered a stimulating approach for reducing bacterial pathogenicity and resistance. Although several QS inhibitors (QSIs) have been discovered so far their clinical use remains distant. This problem can be circumvented by searching for QSI among drugs already approved for the treatment of different diseases. In this context, antibiotics have earned special attention. Whereas at high concentrations antibiotics exert a killing effect, at lower concentrations they may act as signaling molecules and as such can modulate gene expression. In this study, the antibiotic furvina was shown to be able to cause inhibition of the 3-oxo-C12-HSL-dependent QS system of Pseudomonas aeruginosa. Furvina interacts with the LasI/LasR system. The data were validated by modeling studies. Furvina can also reduce biofilm formation and decrease the production of QS-controlled virulence factors.     

Quorum-Sensing Genes in Pseudomonas aeruginosa Biofilms: Their Role and Expression Patterns

Acylated homoserine lactone molecules are used by a number of gram-negative bacteria to regulate cell density-dependent gene expression by a mechanism known as quorum sensing (QS). In Pseudomonas aeruginosa, QS or cell-to-cell signaling controls expression of a number of virulence factors, as well as biofilm differentiation. In this study, we investigated the role played by the las and rhl QS systems during the early stages of static biofilm formation when cells are adhering to a surface and forming microcolonies. These studies revealed a marked difference in biofilm formation between the PAO1 parent and the QS mutants when glucose, but not citrate, was used as the sole carbon source. To further elucidate the contribution of lasI and rhlI to biofilm maturation, we utilized fusions to unstable green fluorescent protein in concert with confocal microscopy to perform real-time temporal and spatial studies of these genes in a flowing environment. During the course of 8-day biofilm development, lasI expression was found to progressively decrease over time. Conversely, rhlI expression remained steady throughout biofilm development but occurred in a lower percentage of cells. Spatial analysis revealed that lasI and rhlI were maximally expressed in cells located at the substratum and that expression decreased with increasing biofilm height. Because QS was shown previously to be involved in biofilm differentiation, these findings have important implications for the design of biofilm prevention and eradication strategies.     

Insight in the quorum sensing-driven lifestyle of the non-pathogenic Agrobacterium tumefaciens 6N2 and the interactions with the yeast Meyerozyma guilliermondii

Agrobacterium tumefaciens is considered a prominent phytopathogen, though most isolates are nonpathogenic. Agrobacteria can inhabit plant tissues interacting with other microorganisms. Yeasts are likewise part of these communities. We analyzed the quorum sensing (QS) systems of A. tumefaciens strain 6N2, and its relevance for the interaction with the yeast Meyerozyma guilliermondii, both sugarcane endophytes. We show that strain 6N2 is nonpathogenic, produces OHC8-HSL, OHC10-HSL, OC12-HSL and OHC12-HSL as QS signals, and possesses a complex QS architecture, with one truncated, two complete systems, and three additional QS-signal receptors. A proteomic approach showed differences in QS-regulated proteins between pure (64 proteins) and dual (33 proteins) cultures. Seven proteins were consistently regulated by quorum sensing in pure and dual cultures. M. guilliermondii proteins influenced by QS activity were also evaluated. Several up- and down- regulated proteins differed depending on the bacterial QS. These results show the QS regulation in the bacteria-yeast interactions.     

The effect of Quorum sensing inhibitors on the evolution of CRISPR-based phage immunity in Pseudomonas aeruginosa

Quorum sensing controls the expression of a wide range of important traits in the opportunistic pathogen Pseudomonas aeruginosa, including the expression of virulence genes and its CRISPR-cas immune system, which protects from bacteriophage (phage) infection. This finding has led to the speculation that synthetic quorum sensing inhibitors could be used to limit the evolution of CRISPR immunity during phage therapy. Here we use experimental evolution to explore if and how a quorum sensing inhibitor influences the population and evolutionary dynamics of P. aeruginosa upon phage DMS3vir infection. We find that chemical inhibition of quorum sensing decreases phage adsorption rates due to downregulation of the Type IV pilus, which causes delayed lysis of bacterial cultures and favours the evolution of CRISPR immunity. Our data therefore suggest that inhibiting quorum sensing may reduce rather than improve the therapeutic efficacy of pilus-specific phage, and this is likely a general feature when phage receptors are positively regulated by quorum sensing.Subject terms: Microbiology, Antimicrobials     

AiiA lactonase disrupts N-acylhomoserine lactone and attenuates quorum-sensing-related virulence in Pectobacterium carotovorum EMPCC

Enzymatic degradation of N-acyl homoserine lactone (NAHL) was found to interfere with the quorum sensing (QS) system and related functions in several soil bacteria. In this research, the NAHL lactonase gene aiiA was amplified using aiiA-7F/aiiA7R PCR primers from the quorum sensing inhibitor rhizobacterium Bacillus sp. strain DMS133, and cloned. The plasmid pME7075, carrying the DMS133 aiiA gene under the constitutive lac promoter, was introduced into the plant pathogen Pectobacterium carotovorum EMPCC, creating strain EMPCC/aiiA. Heterologous expression of the DMS133 aiiA gene in EMPCC severely reduced the accumulation of the NAHL throughout growth, and completely prevented pigmentation of the CV026 bioreporter strain. Virulence analysis revealed that the P. carotovorum strain EMPCC/aiiA expressing AiiA lactonase had drastically reduced tissue maceration activity compared with the wild type EMPCC strain. These results provide evidence that AiiA plays an important role in the quorum quenching ability of Bacillus sp. DMS133 whose AHL degradation capacity was investigated previously. In addition, the communication signal-inactivation approach represents a promising strategy for the prevention of diseases in which virulence is regulated by QS signal molecules.     

Quorum sensing inhibitors: An overview

Excessive and indiscriminate use of antibiotics to treat bacterial infections has lead to the emergence of multiple drug resistant strains. Most infectious diseases are caused by bacteria which proliferate within quorum sensing (QS) mediated biofilms. Efforts to disrupt biofilms have enabled the identification of bioactive molecules produced by prokaryotes and eukaryotes. These molecules act primarily by quenching the QS system. The phenomenon is also termed as quorum quenching (QQ). In addition, synthetic compounds have also been found to be effective in QQ. This review focuses primarily on natural and synthetic quorum sensing inhibitors (QSIs) with the potential for treating bacterial infections. It has been opined that the most versatile prokaryotes to produce QSI are likely to be those, which are generally regarded as safe. Among the eukaryotes, certain legumes and traditional medicinal plants are likely to act as QSIs. Such findings are likely to lead to efficient treatments with much lower doses of drugs especially antibiotics than required at present.     

Inhibitory effects of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) on acyl-homoserine lactone-mediated virulence factor production and biofilm formation in Pseudomonas aeruginosa PAO1

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), a non-halogenated furanone found in a variety of fruits, has been shown to have antimicrobial activity. However, few studies have focused on its inhibitory effect on bacterial quorum sensing (QS) at levels below the non-inhibitory concentration. In this study, 0.1 μM HDMF decreased the production of QS signal molecules and inhibited QS-controlled biofilm formation by Pseudomonas aeruginosa PAO1 without causing growth inhibition. In the presence of 0.1 and 1.0 μM HDMF, biofilm production by PAO1 was reduced by 27.8 and 42.6%, respectively, compared to that by untreated control cells. HDMF (1.0 μM) also significantly affected virulence factor expression (regulated by the las, rhl, and pqs system), resulting in a significant reduction in the production of LasA protease (53.8%), rhamnolipid (40.9%), and pyocyanin (51.4%). This HDMF-dependent inhibition of virulence factor expression was overcome by increasing the levels of two QS signal molecules of P. aeruginosa, N-(3-oxo-dodecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, suggesting reversible competitive inhibition between HDMF and these molecules. The results of this study indicate that HDMF has great potential as an inhibitor of QS, and that it may be of value as a therapeutic agent and in biofilm control, without increasing selective pressure for resistance development.