Degradation of N-Acyl Homoserine Lactone Quorum Sensing Signals by Bacillus thuringiensis AHL Lactonase

Bacterial cells rely on signaling molecules to communicate with others from the same species and induce certain genes in a process known as quorum sensing (QS). A common molecule is N-acyl homoserine lactone (AHL) which is responsible for the expression of virulence and other factors that allow the organisms to compete in a given environment. On the other hand, other bacteria produce certain enzymes such as AHL-lactonase that break down AHL molecules and prevent gene expression of these factors. The aim of this work was to examine the level of degradation of AHL molecules by AHL-lactonase in 62 Bacillus thuringiensis (Bt) strains isolated from Middle Tennessee, Mississippi, and Alabama. N-hexanoyl-homoserine lactone (C6-HSL) and N-3-oxo-hexanoyl homoserine lactone (3-oxo-C6-HSL), which cause Chromobacterium violaceum (CV026) to produce a purple pigment were tested at different concentrations to view the Bt lactonase activity. In addition, PCR was used to test for the presence of the lactonase gene. The results showed that among the 62 Bt strains, there were 58 that possessed the AHL-lactonase (aiiA) gene and 48 strains were able to degrade C6-HSL. At high concentrations of AHL, only 13 strains were able to completely degrade C6-HSL. In addition, degradation of 3-oxo-C6-HSL was weak compared to C6-HSL. The results also revealed that AHL lactonase was thermostable, and it was concluded that the level of degradation varies in Bt strains. Only 13 of the strains studied have potent inhibitory activity against C6-HSL, which may be good to be used in field applications to control agricultural pest.

P_(luxI)mutants with different promoting period and their application for quorum sensing regulated protein expression

Quorum sensing(QS)system can dynamically control the expression of proteins along with the cell growth.The promoting period of QS system has been little focused on until now.In this study,a self-induced dynamic regulated expression(SIDRE)system was constructed in Escherichia coli.To enable the system suitable for the expression of enzymes,promoter engineering was used to obtain P_(luxI)mutants.To test the SIDRE system,alginate lyase AL493 and esterase Est7 were used as target protein for expression.The enzyme activity of alginate lyase and esterase reached 96.38%and 106.71%of the control strains containing the T7 promoter.In high-density fermentation,the activity of alginate lyase expressed by the SIDRE system with P_(luxI)(T-38C)as promoter was 4.34-fold of that expressed by the T7 promoter.Therefore,the P_(luxI)mutants with different promoting periods and/or different strengths show great potential in both laboratory and industrial scale for protein expression.

Anti-Biofilm, Anti-Quorum Sensing, and Anti-Proliferative Activities of Methanolic and Aqueous Roots Extracts of Carica papaya L. and Cocos nucifera L.

Objective: This study focused on the antibacterial and anti-proliferative activity of extracts from Carica papaya and Cocos nucifera roots. Methodology: The minimum inhibitory concentration and the minimum bactericidal concentration of the extracts on Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans, and Staphylococcus aureus were deduced by the microdilution method. The anti-biofilm activity was determined on all four strains and anti-quorum sensing activity by inhibition of violacein production in Chromobacterium violaceum. Anti-proliferative activity on prostate cultured cancer cells was evaluated by MTT assay. Sterols and triterpenes were also assayed in this study. Results: The methanolic extract of Carica papaya showed the best anti-biofilm effect with a percentage inhibition of 66.10 ± 1.79. The methanolic extract of Cocos nucifera had the strongest inhibition on the production of quorum sensing (61.42 ± 0.28). In addition, the methanolic extract of Cocos nucifera roots showed the best cytotoxic effect on prostate cancer LNCaP cell lines (IC50 = 26.98 ± 2.6 μg/mL) and Carica papaya on the PC-3 lines (IC50 = 127.20 ± 5.99 μg/mL). The extracts were also rich in triterpenes and sterols. Conclusion: This study provides support for the ethnomedical use of Carica papaya and Cocos nucifera roots as an antimicrobial and anticancer.

Role of quorum sensing in bacterial infections

Quorum sensing(QS) is cell communication that is widely used by bacterial pathogens to coordinate the expression of several collective traits, including the production of multiple virulence factors, biofilm formation, and swarming motility once a population threshold is reached. Several lines of evidence indicate that QS enhances virulence of bacterial pathogens in animal models as well as in human infections; however, its relative importance for bacterial pathogenesis is still incomplete. In this review, we discuss the present evidence from in vitro and in vivo experiments in animal models, as well as from clinical studies, that link QS systems with human infections. We focus on two major QS bacterial models, the opportunistic Gram negative bacteria Pseudomonas aeruginosa and the Gram positive Staphylococcus aureus, which are also two of the main agents responsible of nosocomial and wound infections. In addition, QS communication systems in other bacterial, eukaryotic pathogens, and even immune and cancer cells are also reviewed, and finally, the new approaches proposed to combat bacterial infections by the attenuation of their QS communication systems and virulence are also discussed.

Initial detection of the quorum sensing autoinducer activity in the rumen of goats in vivo and in vitro

Quorum sensing（QS） is a type of microbe-microbe communication system that is widespread among the microbial world, particularly among microorganisms that are symbiotic with plants and animals. Thereby, the cell-cell signalling is likely to occur in an anaerobic rumen environment, which is a complex microbial ecosystem. In this study, using six ruminally fistulated Liuyang black goats as experimental animals, we aimed to detect the activity of quorum sensing autoinducers（AI） both in vivo and in vitro and to clone the lux S gene that encoded autoinducer-2（AI-2） synthase of microbial samples that were collected from the rumen of goats. Neutral detergent fiber（NDF） and soluble starch were the two types of substrates that were used for in vitro fermentation. The fermented fluid samples were collected at 0, 2, 4, 6, 8, 12, 24, 36, and 48 h of incubation. The acyl-homoserine lactones（AHLs） activity was determined using gas chromatography-mass spectrometer（GC-MS） analysis. However, none of the rumen fluid extracts that were collected from the goat rumen showed the same or similar fragmentation pattern to AHLs standards. Meanwhile, the AI-2 activity, assayed using a Vibrio harveyi BB170 bioassay, was negative in all samples that were collected from the goat rumen and from in vitro fermentation fluids. Our results indicated that the activities of AHLs and AI-2 were not detected in the ruminal contents from six goats and in ruminal fluids obtained from in vitro fermentation at different sampling time-points. However, the homologues of lux S in Prevotella ruminicola were cloned from in vivo and in vitro ruminal fluids. We concluded that AHLs and AI-2 could not be detected in in vivo and in vitro ruminal fluids of goats using the current detection techniques under current dietary conditions. However, the microbes that inhabited the goat rumen had the potential ability to secrete AI-2 signaling molecules and to communicate with each other via AI-2-mediated QS because of the presence of lux S.

Gossip in the gut: Quorum sensing, a new player in the hostmicrobiota interactions

Bacteria are known to communicate with each other and regulate their activities in social networks by secreting and sensing signaling molecules called autoinducers,a process known as quorum sensing(QS).This is a growing area of research in which we are expanding our understanding of how bacteria collectively modify their behavior but are also involved in the crosstalk between the host and gut microbiome.This is particularly relevant in the case of pathologies associated with dysbiosis or disorders of the intestinal ecosystem.This review will examine the different QS systems and the evidence for their presence in the intestinal ecosystem.We will also provide clues on the role of QS molecules that may exert,directly or indirectly through their bacterial gossip,an influence on intestinal epithelial barrier function,intestinal inflammation,and intestinal carcinogenesis.This review aims to provide evidence on the role of QS molecules in gut physiology and the potential shared by this new player.Better understanding the impact of intestinal bacterial social networks and ultimately developing new therapeutic strategies to control intestinal disorders remains a challenge that needs to be addressed in the future.

Anti-quorum sensing and anti-biofilm formation activities of plant extracts from South Korea

Objective: To investigate anti-quorum sensing(anti-QS) and anti-biofilm formation(antiBF) activities of the ethanol extracts of 388 plants. Methods: The anti-QS activity of the plant extracts was evaluated by disc-diffusion assays using the bio-reporter strain, Chromobacterium violaceum CV017. Pseudomonas aeruginosa PAO1, Yersinia enterocolitica ATCC 9610, and Agrobacterium tumefaciens C58, which possess QS systems, were used to evaluate the antiBF activity of the plant extracts. Results: Among 388 plant extracts, the Cornus controversa(C. controversa) and Cynanchum wilfordii extracts exhibited the strongest anti-QS activity. The C. controversa extract exhibited anti-BF activity against Pseudomonas aeruginosa, Yersinia enterocolitica and Agrobacterium tumefaciens, whereas the Cynanchum wilfordii extract exhibited no anti-BF activity against Pseudomonas aeruginosa. In addition, the C. controversa extract suppressed soft rot of cabbage. Conclusions: The C. controversa extract inhibits bacterial QS and BF, and is capable of controlling soft rot. Therefore, this extract has potential for the prevention and treatment of bacterial infections and for the development of alternatives to antibiotics.

Screening strategies for quorum sensing inhibitors in combating bacterial infections

Interference with quorum sensing(QS)represents an antivirulence strategy with a significant promise for the treatment of bacterial infections and a new approach to restoring antibiotic tolerance.Over the past two decades,a novel series of studies have reported that quorum quenching approaches and the discovery of quorum sensing inhibitors(QSIs)have a strong impact on the discovery of anti-infective drugs against various types of bacteria.The discovery of QSI was demonstrated to be an appropriate strategy to expand the anti-infective therapeutic approaches to complement classical antibiotics and antimicrobial agents.For the discovery of QSIs,diverse approaches exist and develop in-step with the scale of screening as well as specific QS systems.This review highlights the latest findings in strategies and methodologies for QSI screening,involving activity-based screening with bioassays,chemical methods to seek bacterial QS pathways for QSI discovery,virtual screening for QSI screening,and other potential tools for interpreting QS signaling,which are innovative routes for future efforts to discover additional QSIs to combat bacterial infections.

Monooxygenase Gene from Acinetobacter sp. C42 Inactivates the Acyl Homoserine Lactone Quorum Sensing Signal

[ Objective] This study aimed to investigate the function of aliD gene in the inactivation of AHLs. [ Method ] A bacterial isolate, Acinetobacter sp. CA2 from soil, is capable of inactivation of AHLs. A gene designed as aliD, which is responsible for AHL-quenching activity and exhibits high similarity with Mo- nooxygenase genes, was cloned from the genomic library of Acinetobacter sp. CA2. [ Result ] The aliD gene in-frame deletion mutant, CA2 AliD, impaired its AHLs inactivating function when mixed with N-（3-oxooctanoyl） -L-homosefine lactone （30C8-HSL）. Expression of AliD in plant pathogenic bacterium Pectobacterium ca- rotovorum subsp, carotovorum Z3-3 significantly reduced the AHLs production and the extracellular pectolytic enzyme activities, and attenuated soft rot disease symptoms on the plants tested, including potato, Chinese cabbage, radish and cabbage. [ Conclusion ] Our study suggests that the aliD gene complemented strain CA2-AliD showes a similar AHLs inactivating function.

Biofilm Formation by Marine Cobetia marina alex and Pseudoaltero­monas spp: Development and Detection of Quorum Sensing N-Acyl Homoserine Lactones (AHLs) Molecules

Surfaces submerged in seawater are colonized by various microorganisms,resulting in the formation of heterogenic marine biofilms.This work aims to evaluate the biofilm formation by Cobetia marina alex and doing a comparative study between this promising strain with the two bacterial strains isolated previously from the Mediterranean seawater,Alexandria,Egypt.Three strains;Cobetia marina alex,Pseudoalteromonas sp.alex,and Pseudoalteromonas prydzensis alex were screened for biofilm formation using the crystal violet(CV)quantification method in a single culture.The values of biofilm formed were OD600=3.0,2.7,and 2.6,respectively leading to their selection for further evaluation.However,factors affecting biofilm formation by C.marina alex were investigated.Biofilm formation was evaluated in single and multispecies consortia.Synergistic and antagonistic interactions proved in this work lead to the belief that these bacteria have the capability to produce some interesting signal molecules N-acyl Homoserine Lactones(AHLs).

Potential of polyphenols in curbing quorum sensing and biofilm formation in Gramnegative pathogens

Polyphenols are the secondary metabolic products of plants and are considered as active constituents to possess therapeutic effects.To date,a vast number of scientific literature addressed the potential of polyphenols as bio-efficient compounds owing to their structural diversity.Due to the presence of several hydroxyl groups,they are metabolized quickly due to conjugation reaction and thus,readily produce toxic metabolites as a defense material against many pathogens,reflecting their safety strategy.This review focuses on the anti-quorum sensing and biofilm inhibition activity of polyphenols,which display their potential to treat bacterial infections by combating the virulence caused by pathogenic agents.Thus,for mitigating quorum sensing-controlled pathogenesis,the use of polyphenol-based phytochemicals holds immense potential to cure infections.The application of polyphenol as sensitizing agent/adjuvant therapeutics which act in synergism with antibiotics is highly remarkable.

Quantitative modeling of bacterial quorum sensing dynamics in time and space

Quorum sensing (QS) refers to the cell communication through signaling molecules that regulate many important biological functions of bacteria by monitoring their population density. Although a wide spectrum of studies on the QS system mechanisms have been carried out in experiments, mathematical modeling to explore the QS system has become a powerful approach as well. In this paper, we review the research progress of network modeling in bacterial QS to capture the system's underlying mechanisms. There are four types of QS system models for bacteria: the Gram-negative QS system model, the Gram-positive QS system model, the model for both Gram-negative and Gram-positive QS system, and the synthetic QS system model. These QS system models are mostly described by the ordinary differential equations (ODE) or partial differential equations (PDE) to study the changes of signaling molecule dynamics in time and space and the cell population density variations. Besides the deterministic simulations, the stochastic modeling approaches have also been introduced to discuss the noise effects on kinetics in QS systems. Taken together, these current modeling efforts advance our understanding of the QS system by providing systematic and quantitative dynamics description, which can hardly be obtained in experiments.

Quality evaluation of synthetic quorum sensing peptides used in R&D

Peptides are becoming an important class of molecules in the pharmaceutical field. Closely related peptide-impurities in peptides are inherent to the synthesis approach and have demonstrated to potentially mask biomedical experimental results. Quorum sensing peptides are attracting high interest in R＆D and therefore a representative set of quorum sensing peptides, with a requested purity of at least 95.0%, was evaluated for their purity and nature of related impurities. In-house quality control （QC） revealed a large discrepancy between the purity levels as stated on the supplier＇s certificate of analysis and our QC results. By using our QC analysis flowchart, we demonstrated that only 44.0% of the peptides met the required purity. The main compound of one sample was even found to have a different structure compared to the desired peptide. We also found that the majority of the related impurities were lacking amino acid（s） in the desired peptide sequence. Relying on the certificates of analysis as provided by the supplier might have serious consequences for peptide research, and peptide-researchers should implement and maintain a thorough in-house QC.

Research Progress of Quorum Sensing in Staphylococcus aureus

Quorum sensing refers to the phenomenon that bacteria sense signal molecules in the environment and regulate a series of genes.At present,the known quorum sensing systems in Staphylococcus aureus are the Agr system and the LuxS/AI-2 system.They will be activated when the bacterial concentration is equal to or greater than 107/ml,and by regulating the corresponding genes,bacteria can indirectly or directly regulate the production and degradation of biofilms,the secretion of bacterial toxins and the growth of bacteria.In this paper,we summarized the research progress of quorum sensing in S.aureus by consulting relevant literatures at home and abroad on quorum sensing in S.aureus,so as to find a new direction for the future research on S.aureus.

Catechol-derived Inhibitors against Quorum Sensing from Retiboletus kauffmanii

[Objectives]This study aimed to screen anti-quorum sensing(QS)activity from the constituent of Retiboletus kauffmanii fruit body.[Methods]Chromatographic technology was used to isolate compounds,while well diffusion assay was applied to screen anti-quorum sensing activity.[Results]12 phenolic compounds were purified from extracts,three catechol-derived compounds at sub-inhibitory concentrations were found to inhibit the production of violacein in Chromobacterium violaceum for the first time.[Conclusions]Three catechol analogs isolated from R.kauffmanii fruit body extract were indicated as quorum sensing inhibitors against C.violaceum.

Genetic and biochemical aspects of quorum sensing in the bacterial lifestyle and pathogenesis

Quorum sensing is the biochemical communication system within bacterial cells which may instigate their virulence factors,and hence,is imperative for pathogenesis.Such a system optimizes metabolic and behavioral activities played by the densely populated bacterial community as well as the production of the concomitant response against diffusible or secreted signals.Thus,the multi-layered signal transduction networks of quorum sensing help bacteria acclimatize to the disadvantages by increasing their confrontation ability aided by biofilm formation.Previous studies unraveled the impact of quorum sensing in the microbial interactions both within a single species and between several species.The development of the synthetic ecological models has added advantages in the studies of microbial sociability in natural habitats as well as aided in drug designing.Current review highlighted the up-to-date knowledge on the basic mechanisms of quorum sensing and its influence to accelerate the bacterial resistance against different environmental stimuli or synthetic drugs along with the factors affecting the quorum sensing system,and the harmful role of quorum sensing in food spoilage.The information gathered in this review would be beneficial to understand the metabolic cross talk as well as the virulence traits of the pathogenic microorganisms in course of their pathogenesis augmented by quorum sensing.

RsaL is a self-regulatory switch that controls alternative biosynthesis of two AHL-type quorum sensing signals in Pseudomonas aeruginosa PA1201

Pseudomonas aeruginosa is a ubiquitous and metabolically versatile microorganism naturally found in soil and water.It is also an opportunistic pathogen in plants,insects,animals,and humans.In response to increasing cell density,P.aeruginosa uses two acylhomoserine lactone(AHL)quorum-sensing(QS)signals(i.e.,N-3-oxo-dodecanoyl homoserine lactone[3-oxo-C12-HSL]and Nbutanoyl-homoserine lactone[C4-HsL]),which regulate the expression of hundreds of genes.However,how the biosynthesis of these two QS signals is coordinated remains unknown.We studied the regulation of these two QS signals in the rhizosphere strain PA1201.PA1201 sequentially produced 3-oxo-C12-HSL and C4-HSL at the early and late growth stages,respectively.The highest 3-oxo-C12-HSL-dependent elastase activity was observed at the early stage,while the highest C4-HSL-dependent rhamnolipid production was observed at the late stage.The atypical regulator RsaL played a pivotal role in coordinating 3-oxo-C12-HSL and C4-HSL biosynthesis and QS-associated virulence.RsaL repressed las/transcription by binding the-10 and-35 boxes of the lasl promoter.In contrast,RsaL activated rhll transcription by binding the region encoding the 5'-untranslated region of the rhll mRNA.Further,RsaL repressed its own expression by binding a nucleotide motif located in the-35 box of the rsaL promoter.Thus,RsaL acts as a molecular switch that coordinates the sequential biosynthesis of AHL QS signals and differential virulence in PA1201.Finally,C4-HSL activation by RsaL was independent of the Las and Pseudomonas quinolone signal(PQS)QS signaling systems.Therefore,we propose a new model of the QS regulatory network in PA1201,in which RsaL represents a superior player acting at the top of the hierarchy.

Quorum sensing-based interactions among drugs,microbes,and diseases

Many diseases and health conditions are closely related to various microbes,which participate in complex interactions with diverse drugs;nonetheless,the detailed targets of such drugs remain to be elucidated.Many existing studies have reported causal associations among drugs,gut microbes,or diseases,calling for a workflow to reveal their intricate interactions.In this study,we developed a systematic workflow comprising three modules to construct a Quorum Sensing-based Drug-Microbe-Disease(QSDMD)database(http://www.qsdmd.lbci.net/),which includes diverse interactions for more than 8,000 drugs,163 microbes,and 42 common diseases.Potential interactions between microbes and more than 8,000 drugs have been systematically studied by targeting microbial QS receptors combined with a docking-based virtual screening technique and in vitro experimental validations.Furthermore,we have constructed a QS-based drug-receptor interaction network,proposed a systematic framework including various drug-receptor-microbe-disease connections,and mapped a paradigmatic circular interaction network based on the QS-DMD,which can provide the underlying QS-based mechanisms for the reported causal associations.The QS-DMD will promote an understanding of personalized medicine and the development of potential therapies for diverse diseases.This work contributes to a paradigm for the construction of a molecule-receptor-microbe-disease interaction network for human health that may form one of the key knowledge maps of precision medicine in the future.

The hierarchy quorum sensing networ in Pseudomonas aeruginosa

Pseudomonas aeruginosa causes severe and persistent infections in immune compromised individuals and cystic fibrosis sufferers. The infection is hard to eradi- cate as P. aeruginosa has developed strong resistance to most conventional antibiotics. The problem is further compounded by the ability of the pathogen to form biofilm matrix, which provides bacterial cells a protected environment withstanding various stresses including antibiotics. Quorum sensing （QS）, a cell density-based intercellular communication system, which plays a key role in regulation of the bacterial virulence and biofilm formation, could be a promising target for developing new strategies against P. aeruginosa infection. The QS network of P. aeruginosa is organized in a multi-layered hierarchy consisting of at least four interconnected signaling mechanisms. Evidence is accumulating that the QS regulatory network not only responds to bacte- rial population changes but also could react to envi- ronmental stress cues. This plasticity should be taken into consideration during exploration and development of anti-QS therapeutics.

Effects of quorum sensing autoinducer degradation gene on virulence and biofilm formation of Pseudomonas aeruginosa

The aiiA gene from Bacillus thuringiensis was cloned into the Pseudomonas/E. coli shuttle vector and transformed into Pseudomonas aeruginosa strain PAO1. Western blotting showed that the AiiA protein was expressed in PAO1. After induction by IPTG for 6 h and 18 h, expression of the aiiA gene in PAO1 completely degraded the quorum sensing autoinducers N-acylhomoserine lactones (AHLs): N-oxododecanoyl-L-homoserine lactone (OdDHL) and N-butyryl-L-homoserine lactone (BHL). The re- duced amount of AHLs in PAO1 was also correlated with decreased expression and production of several virulence factors such as elastase and pyocyanin. AiiA expression also influenced bacterial swarming motility. Most importantly, our studies indicated that aiiA played significant roles in P. aeruginosa biofilm formation and dispersion, as observed by the differences of the biofilm formation on liquid and solid surfaces, and biofilm structures under a scanning electron microscope.