Biosynthesis of Glycomonoterpenes to Attenuate Quorum Sensing Associated Virulence in Bacteria

The acquisition of multidrug resistance in bacteria has become a bigger threat of late, mainly due to the bacterial signaling phenomenon, quorum sensing (QS). QS, among a population of bacteria, initiates the formation of biofilms and offers myriad advantages to bacteria. Burgeoning antibiotic resistance in biofilm-producing bacteria has motivated efforts toward finding new alternatives to these traditional antimicrobials. In the present study, we report the increased solubility and additional quorum quenching as well as biofilm disruption activity of glyco-derivatives of monoterpenes (citral and citronellal). Glycomonoterpenes of citral and citronellal were synthesized via conjugation of the monoterpenes with glucose by the non-pathogenic yeast Candida bombicola (ATCC 22214). Structural elucidation of newly synthesized glycomonoterpenes showed that one synthesized using citronellal contains three major lactonic forms with molecular weight 492.43, 473.47, and 330.39 Da whereas the one produced using citral has an acidic form with molecular weight 389.33 and 346.23 Da. The glycomonoterpenes were able to individually inhibit QS, mediated through various medium-chain and long-chain N-acyl homoserine lactones (AHLs). These new compounds are interesting additions to the known range of quorum sensing inhibitors (QSIs) and could be further explored for potential clinical applications.     

Homogentisic acid γ-lactone suppresses the virulence factors of Pseudomonas aeruginosa by quenching its quorum sensing signal molecules

Homogentisic acid γ-lactone exhibited excellent anti-quorum sensing (QS) and anti-biofilm activities against Pseudomonas aeruginosa. Moreover, it suppressed the QS-dependent virulence factors in P. aeruginosa by quenching its QS signal molecules.     

Inhibition of the Quorum Sensing System,Elastase Production and Biofilm Formation in Pseudomonas aeruginosa by Psammaplin A and Bisaprasin

Natural products derived from marine sponges have exhibited bioactivity and, in some cases, serve as potent quorum sensing inhibitory agents that prevent biofilm formation and attenuate virulence factor expression by pathogenic microorganisms. In this study, the inhibitory activity of the psammaplin-type compounds, psammaplin A (1) and bisaprasin (2), isolated from the marine sponge, Aplysinella rhax, are evaluated in quorum sensing inhibitory assays based on the Pseudomonas aeruginosa PAO1 lasB-gfp(ASV) and rhlA-gfp(ASV) biosensor strains. The results indicate that psammaplin A (1) showed moderate inhibition on lasB-gfp expression, but significantly inhibited the QS-gene promoter, rhlA-gfp, with IC₅₀ values at 14.02 μM and 4.99 μM, respectively. In contrast, bisaprasin (2) displayed significant florescence inhibition in both biosensors, PAO1 lasB-gfp and rhlA-gfp, with IC₅₀ values at 3.53 μM and 2.41 μM, respectively. Preliminary analysis suggested the importance of the bromotyrosine and oxime functionalities for QSI activity in these molecules. In addition, psammaplin A and bisaprasin downregulated elastase expression as determined by the standard enzymatic elastase assay, although greater reduction in elastase production was observed with 1 at 50 μM and 100 μM. Furthermore, the study revealed that bisaprasin (2) reduced biofilm formation in P. aeruginosa.     

Microfluidic approaches to bacterial biofilm formation

Bacterial biofilms-aggregations of bacterial cells and extracellular polymeric substrates (EPS)-are an important subject of research in the fields of biology and medical science. Under aquatic conditions, bacterial cells form biofilms as a mechanism for improving survival and dispersion. In this review, we discuss bacterial biofilm development as a structurally and dynamically complex biological system and propose microfluidic approaches for the study of bacterial biofilms. Biofilms develop through a series of steps as bacteria interact with their environment. Gene expression and environmental conditions, including surface properties, hydrodynamic conditions, quorum sensing signals, and the characteristics of the medium, can have positive or negative influences on bacterial biofilm formation. The influences of each factor and the combined effects of multiple factors may be addressed using microfluidic approaches, which provide a promising means for controlling the hydrodynamic conditions, establishing stable chemical gradients, performing measurement in a high-throughput manner, providing real-time monitoring, and providing in vivo-like in vitro culture devices. An increased understanding of biofilms derived from microfluidic approaches may be relevant to improving our understanding of the contributions of determinants to bacterial biofilm development.     

Synthesis of the quorum sensing molecule Diffusible Signal Factor using the alkyne zipper reaction

The development of a concise synthesis of the quorum sensing molecule Diffusible Signal Factor is described. This route exploits an alkyne zipper reaction to form a key terminal alkyne intermediate. The chemistry outlined here may also be applied to the preparation of cis-unsaturated analogues of Diffusible Signal Factor.     

Inhibition of Quorum Sensing,Motility and Biofilm Formation of Pseudomonas aeruginosa by Copper Oxide Nanostructures

Quorum sensing (QS) is the communication between bacterial cells governed by their population density and regulated by the genes controlling virulence factors and biofilm formation. Multiple mechanisms of biofilms are resistive to antimicrobial chemotherapy; therefore novel strategies are required to overcome its limitations. Here, we report the effect of various copper oxide nanostructures (CuO-NSs) on quorum sensing inhibition. The two-dimensional CuO-NSs such as interlaced nanodiscs, nanodiscs and leaf-shaped nanosheets are prepared via a simple chemical method. The Quorum sensing inhibition (QSI) activity of all the CuO-NS are examined using reporter strain Chromobacterium violaceum CV026 and Escherichia coli pSB1142. We found that the CuO-interlaced nanodisc structures exhibit better QSI activity than nanodiscs and leaf-shaped sheets. The interlaced nanodisc structures are inhibited various long-chain N-acyl homoserine lactones (AHLs) mediated QS individually and confirmed by other QS-associated phenomena for Pseudomonas aeruginosa, including biofilm inhibition, inhibition of virulence factors such as pyocyanin, protease production and swarming motility. Thus QSI activity of CuO-NSs is solely dependent on specific shape offering large surface area and more active sites. The CuO-NS is effective quorum sensing inhibitors, which has potential clinical applications in the management of P. aeruginosa associated infections.

     

Effect of Cyclodextrins on the Biofilm Formation Capacity of Pseudomonas aeruginosa PAO1

Quorum sensing (QS) is a population-density-dependent communication process of microorganisms to coordinate their activities by producing and detecting low-molecular-weight signal molecules. In pathogenic bacteria, the property controlled by QS is often related to infectivity, e.g., biofilm formation. Molecular encapsulation of the QS signals is an innovative method to prevent the signals binding to the receptors and to attenuate QS. Cyclodextrins (CDs) may form an inclusion complex with the signals, thus reducing the communication (quorum quenching, QQ). A systematic study was performed with α-, β-cyclodextrin, and their random methylated, quaternary amino and polymer derivatives to evaluate and compare their effects on the biofilm formation of Pseudomonas aeruginosa. To examine the concentration-, temperature- and time-dependency of the QQ effect, the CDs were applied at a 0.1–12.5 mM concentration range, and biofilm formation was studied after 6, 24, 48 and 72 h at 22 and 30 °C. According to the results, the QS mechanism was significantly inhibited; the size of the cavity, the structure of the substituents, as well as the monomeric or polymeric character together with the concentration of the CDs have been identified as key influencing factors of biofilm formation. Statistically determined effective concentration values demonstrated outstanding efficiency (higher than 80% inhibition) of α-CD and its random methylated and polymer derivatives both on the short and long term. In summary, the potential value of CDs as inhibitors of QS should be considered since the inhibition of biofilm formation could significantly impact human health and the environment.     

Computational discovery of putative quorum sensing inhibitors against LasR and RhlR receptor proteins of Pseudomonas aeruginosa

Drugs have been discovered in the past mainly either by identification of active components from traditional remedies or by unpredicted discovery. A key motivation for the study of structure based virtual screening is the exploitation of such information to design targeted drugs. In this study, structure based virtual screening was used in search for putative quorum sensing inhibitors (QSI) of Pseudomonas aeruginosa. The virtual screening programme Glide version 5.5 was applied to screen 1,920 natural compounds/drugs against LasR and RhlR receptor proteins of P. aeruginosa. Based on the results of in silico docking analysis, five top ranking compounds namely rosmarinic acid, naringin, chlorogenic acid, morin and mangiferin were subjected to in vitro bioassays against laboratory strain PAO1 and two more antibiotic resistant clinical isolates, P. aeruginosa AS1 (GU447237) and P. aeruginosa AS2 (GU447238). Among the five compounds studied, except mangiferin other four compounds showed significant inhibition in the production of protease, elastase and hemolysin. Further, all the five compounds potentially inhibited the biofilm related behaviours. This interaction study provided promising ligands to inhibit the quorum sensing (QS) mediated virulence factors production in P. aeruginosa.     

Small molecule inhibitors of bacterial quorum sensing and biofilm formation

Bacteria monitor their local population densities using small molecules (or autoinducers) in a process known as quorum sensing. Here, we report a new and efficient synthetic route to naturally occurring bacterial autoinducers [N-acyl l-homoserine lactones (AHLs)] that is readily amenable to the synthesis of analogues. This route has been applied in the first synthesis of a library of non-native AHLs. Evaluation of these compounds in bacterial reporter gene and biofilm assays has revealed a potent set of quorum sensing antagonists. These ligands will serve as valuable new tools to explore the role of quorum sensing in bacterial pathogenesis.     

Localized quorum sensing in Vibrio fischeri

Quorum sensing is almost always regarded as a population density effect in three-dimensional bulk samples of bacteria. Here we create two-dimensional samples of Vibrio fischeri cells adhered onto glass surfaces to examine the effect of local population densities on quorum sensing. This is done by measuring the luminescent response. The 2-D bacterial populations enable us to simultaneously account for time and distance effects on quorum sensing, which were previously very challenging to access in typical three-dimensional bulk samples. Thus, we are able to consider quorum sensing in terms of signal diffusion. A diffusion model of quorum sensing signals guides the experiments and shows that for a given cell spacing (density) and diffusion time there exists a “true quorum”— a number of cells necessary for quorum sensing. We find that quorum sensing can occur locally in 2-D surface samples and is a function of cell population density as well as signal diffusion time.     

Robust routes for the synthesis of N-acylated-l-homoserine lactone (AHL) quorum sensing molecules with high levels of enantiomeric purity

The ready availability of native quorum sensing molecules and related structural analogues is of significant biological interest in the development of methods to manipulate bacterial quorum sensing systems in a useful fashion. In this Letter we report robust routes for the synthesis of a range of N-acylated-l-homoserine lactone (AHL) quorum sensing molecules. Crucially, we have analysed the enantiopurity of the final AHLs and in all cases, excellent levels were observed.     

Design,synthesis, biological evaluation and in silico studies of N-(pyridin-2-yl)-benzamides derivatives as quorum sensing inhibitors

The emergence of bacterial resistance against chemical treatment is a big threat to the efficacy of bacterial infection treatment. One of the major reasons for resistance to antimicrobial agents is growth of microorganisms in biofilm. An alternative treatment by developing novel anti-biofilm agents had led to the concept of quorum sensing (QS) inhibition, which primarily targets QS signaling system by disrupting cell-cell communication. Therefore, this study focuses to develop novel antimicrobial agents which work by QS inhibition and act as anti-biofilm agents against Bacillus Subtilis and Pseudomonas Aeruginosa. In this work, a natural product-like scaffolds from Asinex library were screened and N-pyridin-2-yl-benzamide moiety was chosen to design and synthesize. Synthesized compounds were evaluated for potential anti-biofilm activity for the aforesaid microorganisms and also checked for cell viability assay, where two potent compounds 3a and 3c showed their static biofilm activity to ∼59% and ∼58% at 100 μM, respectively against Bacillus subtilis. These synthesized compounds were investigated for physicochemical parameters and binding mode prediction through molecular modelling tools. The interactions and stability of these compounds showed better affinity towards TasA and LasR proteins from Bacillus subtilis and Pseudomonas aeruginosa, respectively. Furthermore, molecular dynamic simulation for 100 ns was executed in order to appreciate the stability of the protein and ligand complex. The overall results promised that N-pyridin-2-yl-benzamide derivatives can be discovered as a lead in developing potent anti-quorum sensing agents against various bacteria.     

Analysis of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 Biofilm Protein Profile After Exposure to <Emphasis Type="Italic">n</Emphasis>-Butanolic <Emphasis Type="Italic">Cyclamen coum</Emphasis> Extract Alone and in Combination with Ciprofloxacin

Pseudomonas aeruginosa biofilm-related infections are the major cause of premature death in cystic fibrosis patients. Strategies to induce biofilm dispersal are of interest, because of their potential in preventing biofilm-related infections. Our previous work demonstrated that n-butanolic Cyclamen coum extract with ciprofloxacin could eliminate 1- and 3-day-old P. aeruginosa PAO1 biofilms. To gain new insights into the role of C. coum extract and its synergistic effect with ciprofloxacin in eliminating P. aeruginosa PAO1 biofilms, two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry-based protein identification were used. Changes in the bacterial protein expression were analyzed when 3-day-old biofilm cells were exposed to the C. coum extract alone and in combination with ciprofloxacin. Proteins involved in alginate biosynthesis, quorum sensing, adaptation/protection, carbohydrate and amino acid metabolism showed a weaker expression in the C. coum extract-ciprofloxacin-treated biofilm cells compared to those in the untreated cells. Interestingly, the proteome of C. coum extract-ciprofloxacin-treated biofilm revealed more resemblance to the planktonic phenotype than to the biofilm phenotype. It appears that saponin extract in combination with ciprofloxacin causes biofilm disruption due to several mechanisms such as motility induction, cell envelope integrity perturbation, stress protein expression reduction, and more importantly, signal transduction perturbation. In conclusion, exposure to a combination of biofilm dispersal such as saponin extract and antimicrobial agents may offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.     

Nano-enabled Quenching of Bacterial Communications for the Prevention of Biofilm Formation

Biofilm formation is a major threat to industry, the environment and human health. While killing of embedded microbes in biofilms may inevitably lead to the evolution of antimicrobial resistance (AMR), catalytic quenching of bacterial communications by lactonase is a promising antifouling approach. Given the shortcomings of protein enzymes, it is attractive to engineer synthetic materials to mimic the activity of lactonase. Herein, an efficient lactonase-like Zn−N_x−C nanomaterial was synthesized by tuning the coordination environment around zinc atoms to mimic the active domain of lactonase for catalytical interception of bacterial communications in biofilm formation. The Zn−N_x−C material could selectively catalyze 77.5 % hydrolysis of N-acylated-L-homoserine lactone (AHL), a critical bacterial quorum sensing (QS) signal in biofilm construction. Consequently, AHL degradation downregulated the expression of QS-related genes in antibiotic resistant bacteria and significantly prevented biofilm formation. As a proof of concept, Zn−N_x−C-coated iron plates prevented 80.3 % biofouling after a month exposure in river. Overall, our study provides a nano-enabled contactless antifouling insight to avoid AMR evolution by engineering nanomaterials for mimicking the key bacterial enzymes (e.g., lactonase) functioning in biofilm construction.     

In Silico Screening of Quorum Sensing Inhibitor Candidates Obtained by Chemical Similarity Search

Quorum sensing (QS) is a bacterial communication using signal molecules, by which they sense population density of their own species, leading to group behavior such as biofilm formation and virulence. Autoinducer-2 (AI2) is a QS signal molecule universally used by both gram-positive and gram-negative bacteria. Inhibition of QS mediated by AI2 is important for various practical applications, including prevention of gum-disease caused by biofilm formation of oral bacteria. In this research, molecular docking and molecular dynamics (MD) simulations were performed for molecules that are chemically similar to known AI2 inhibitors that might have a potential to be quorum sensing inhibitors. The molecules that form stable complexes with the AI2 receptor protein were found, suggesting that they could be developed as a novel AI2 inhibitors after further in vitro validation. The result suggests that combination of ligand-based drug design and computational methods such as MD simulation, and experimental verification, may lead to development of novel AI inhibitor, with a broad range of practical applications.     

Quorum-sensing systems in staphylococci as therapeutic targets

The staphylococci are an ever-present threat in our world, capable of causing a wide range of infections, and are a persistent presence in the clinical environment. As the number of antimicrobial compounds effective against staphylococci decreases, because of the acquisition and spread of antibiotic resistance, there is a growing need for novel therapeutic molecules. Intra and inter-species communication (quorum sensing) is a biologically significant phenomenon that has been associated with virulence, intracellular survival, and biofilm formation. Quorum sensing molecules of staphylococci and other species (e.g. Pseudomonas aeruginosa) can inhibit virulence factor production and/or growth of staphylococci, leading to the possibility that interference with staphylococcal quorum-sensing systems could be a way of controlling the diverse infections caused by the staphylococci. In this article, we discuss the potential of quorum-sensing systems of staphylococci as therapeutic targets.     

Total synthesis and structural validation of cyclodepsipeptides solonamide A and B

Microorganisms are an attractive source of new natural products with antimicrobial properties, and the marine environment constitutes a prolific resource of bioactive microorganisms. During a global research expedition (Galathea III), two depsipeptides, solonamide A and solonamide B, were isolated from the marine bacterium Photobacterium halotolerance and were found to inhibit virulence gene expression in the serious human pathogen, Staphylococcus aureus. They act by interfering with the agr quorum sensing system and show resemblance to the endogenous S. aureus quorum sensing peptide, autoinducing peptide I (AIP-I). To enable more comprehensive studies, we embarked on the chemical synthesis of solonamides A and B. The key synthetic steps were formation of the (R)-β-hydroxy-fatty-acids by stereo-selective aldol reactions and a cyclative macrolactamization, which proceeded under highly dilute conditions. Thus, the first total syntheses of the solonamides corroborated the originally assigned structures, and by changing the stereochemistry of the auxiliary in the aldol steps we gained access to the natural products as well as their β³-epimers.     

The Derived Components of Gnaphalium hypoleucum DC. Reduce Quorum Sensing of Chromobacterium violaceum

Gnaphalium hypoleucum DC. was first recorded in the Chinese National Pharmacopoeia “Yi Plant Medicine”. There is no detailed report on its main components’ activity in suppressing the quorum sensing activity (QS) of bacteria. Our study aimed to screen the main components in extracts of G. hypoleucum DC. in order to measure their effects on bacterial QS activity and to explore specific quorum sensing mechanisms that are affected by G. hypoleucum DC. extracts. Crude extracts of G. hypoleucum DC. contained significant amounts of two compounds shown to inhibit bacterial QS activity, namely apigenin and luteolin. Apigenin and luteolin in crude extracts of G. hypoleucum DC. showed substantial inhibition of pigment formation, biofilm production, and motility in Chromobacterium violaceum ATCC 12472 compared to the effects of other phytochemicals from G. hypoleucum DC. Apigenin and luteolin exhibited a strong QS inhibitory effect on C. violaceum, interfering with the violacein pigment biosynthesis by downregulating the vioB, vioC, and vioD genes. In the presence of signal molecules, the QS effect is prevented, and the selected compounds can still inhibit the production of the characteristic purple pigment in C. violaceum. Based on qualitative and quantitative research using genomics and bioinformatics, we concluded that apigenin and luteolin in crude extracts of G. hypoleucum DC can interfere with the generation of QS in C. violaceum by downregulating the vioB, vioC, and vioD genes. Indeed, G. hypoleucum DC. is used for the treatment of bacterial infections, and this research provides new ideas and potential alternative uses for medicinal plants.