Bacterial quorum sensing and interference by naturally occurring biomimics

Bacteria are able to coordinate gene expression as a community through the secretion and detection of signalling molecules so that the members of the community can simultaneously express specific behaviours. This mechanism of regulation of behaviour appears to be a key trait for adaptation to specific environments and has been shown to regulate a variety of important phenotypes, from virulence factor production to biofilm formation to symbiosis related behaviours such as bioluminescence. The ability to communicate and communally regulate gene expression is hypothesised to have evolved as a way for organisms to delay expression of phenotypes until numerical supremacy is reached. For example, in the case of infection, if an invading microorganism were to express virulence factors too early, the host may be able to mount a successful defence and repel the invaders. There is growing evidence that bacterial quorum sensing (QS) systems are involved in cross-kingdom signalling with eukaryotic organisms and that eukaryotes are capable of actively responding to bacteria in their environment by detecting and acting upon the presence of these signalling molecules. Likewise, eukaryotes produce compounds that can interfere with QS systems in bacteria by acting as agonists or antagonists. An exciting new field of study, biomimetics, takes inspiration from nature’s models and attempts to design solutions to human problems, and biomimics of QS systems may be one such solution. This article presents the acylated homoserine lactone and autoinducer 2 QS systems in bacteria, the means of intercepting or interfering with bacterial QS systems evolved by eukaryotes, and the rational design of synthetic antagonists. Figure Natural products, furanones, from the red alga Delisea pulchra inhibit the quorum sensing regulated production of violacein by Chromobacterium violaceum “The proof of evolution lies in those adaptations that arise from improbable foundations”—Stephen Jay Gould     

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.     

The quorum-sensing molecule <Emphasis Type="Italic">N</Emphasis>-3-oxododecanoyl homoserine lactone (3OC12-HSL) enhances the host defence by activating human polymorphonuclear neutrophils (PMN)

The P. aeruginosa quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) interacts not only with bacteria, but also with mammalian cells, among others with those of the immune defence system. We focussed on the possible interaction of 3OC12-HSL with human polymorphonuclear neutrophils (PMN), because these cells are the first to enter an infected site. We found that 3OC12-HSL attracts PMN, and up-regulates expression of receptors known to be involved in host defence, including the adhesion proteins CD11b/CD18 and the immunoglobulin receptors CD16 and CD64. Furthermore, the uptake of bacteria (phagocytosis), which is crucial for an efficient defence against infection, was enhanced. Thus, recognising and responding to 3OC12-HSL not only attracts the PMN to the site of a developing biofilm, but also reinforces their defence mechanisms, and hence could be a means to control the infection in an early stage and to prevent biofilm formation.     

Convergence of hormones and autoinducers at the host/pathogen interface

Most living organisms possess sophisticated cell-signaling networks in which lipid-based signals modulate biological effects such as cell differentiation, reproduction and immune responses. Acyl homoserine lactone (AHL) autoinducers are fatty acid-based signaling molecules synthesized by several Gram-negative bacteria that are used to coordinate gene expression in a process termed “quorum sensing” (QS). Recent evidence shows that autoinducers not only control gene expression in bacterial cells, but also alter gene expression in mammalian cells. These alterations include modulation of proinflammatory cytokines and induction of apoptosis. Some of these responses may have deleterious effects on the host’s immune response, thereby leading to increased bacterial pathogenesis. Prokaryotes and eukaryotes have cohabited for approximately two billion years, during which time they have been exposed to each others’ soluble signaling molecules. We postulate that organisms from the different kingdoms of nature have acquired mechanisms to sense and respond to each others signaling molecules, and we have named this process interkingdom signaling. We further propose that autoinducers, which exhibit structural and functional similarities to mammalian lipid-based hormones, are excellent candidates for mediating this interkingdom communication. Here we will compare and contrast bacterial QS systems with eukaryotic endocrine systems, and discuss the mechanisms by which autoinducers may exploit mammalian signal transduction pathways.     

Quorum sensing in <Emphasis Type="Italic">Erwinia</Emphasis> species

The term quorum sensing (QS) refers to the ability of bacteria to regulate gene expression according to the accumulation of signalling molecules that are made by every cell in the population. The erwiniae group of bacteria are often phytopathogens and the expression of a number of their important virulence determinants and secondary metabolites is under QS control. The erwiniae utilise two types of QS signalling molecules: N-acyl homoserine lactones and AI-2-type signalling molecules. Here, we review the regulatory networks involving QS in the soft rot erwiniae.     

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.     

Inhibition of Quorum-Sensing Regulator from Pseudomonas aeruginosa Using a Flavone Derivative

Quorum sensing (QS) is a cell-to-cell communication process that controls bacterial collective behaviors. The QS network regulates and coordinates bacterial virulence factor expression, antibiotic resistance and biofilm formation. Therefore, inhibition of the QS system is an effective strategy to suppress the bacterial virulence. Herein, we identify a phosphate ester derivative of chrysin as a potent QS inhibitor of the human pathogen Pseudomonas aeruginosa (P. aeruginosa) using a designed luciferase reporter assay. In vitro biochemical analysis shows that the chrysin derivative binds to the bacterial QS regulator LasR and abrogates its DNA-binding capability. In particular, the derivative exhibits higher anti-virulence activity compared to the parent molecule. All the results reveal the potential application of flavone derivative as an anti-virulence compound to combat the infectious diseases caused by P. aeruginosa.     

Assessing the Molecular Targets and Mode of Action of Furanone C-30 on Pseudomonas aeruginosa Quorum Sensing

Quorum sensing (QS), a sophisticated system of bacterial communication that depends on population density, is employed by many pathogenic bacteria to regulate virulence. In view of the current reality of antibiotic resistance, it is expected that interfering with QS can address bacterial pathogenicity without stimulating the incidence of resistance. Thus, harnessing QS inhibitors has been considered a promising approach to overriding bacterial infections and combating antibiotic resistance that has become a major threat to public healthcare around the globe. Pseudomonas aeruginosa is one of the most frequent multidrug-resistant bacteria that utilize QS to control virulence. Many natural compounds, including furanones, have demonstrated strong inhibitory effects on several pathogens via blocking or attenuating QS. While the natural furanones show no activity against P. aeruginosa, furanone C-30, a brominated derivative of natural furanone compounds, has been reported to be a potent inhibitor of the QS system of the notorious opportunistic pathogen. In the present study, we assess the molecular targets and mode of action of furanone C-30 on P. aeruginosa QS system. Our results suggest that furanone C-30 binds to LasR at the ligand-binding site but fails to establish interactions with the residues crucial for the protein’s productive conformational changes and folding, thus rendering the protein dysfunctional. We also show that furanone C-30 inhibits RhlR, independent of LasR, suggesting a complex mechanism for the agent beyond what is known to date.     

Inhibition of Swarming motility of Pseudomonas aeruginosa by Methanol extracts of Alpinia officinarum Hance. and Cinnamomum tamala T. Nees and Eberm

Bacterial drug resistance is a challenge in clinical settings, especially in countries like India. Hence, discovery of novel alternative therapeutics has become a necessity in the fight against drug resistance. Compounds that inhibit bacterial virulence properties form new therapeutic alternatives. Pseudomonas aeruginosa is an opportunistic, nosocomial pathogen that infects immune-compromised patients. Swarming motility is an important virulence property of Pseudomonas which aids it in reaching host cells under nutrient limiting conditions. Here, we report the screening of five plant extracts against swarming motility of P. aeruginosa and show that methanol extracts of Alpinia officinarum and Cinnamomum tamala inhibit swarming motility at 5 μg mL^?1 without inhibiting its growth. These extracts did not inhibit swimming and twitching motilities indicating a mode of action specific to swarming pathway. Preliminary experiments indicated that rhamnolipid production was not affected. This study reveals the potential of the two plants in anti-virulence drug discovery.     

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.

     

Electrophoretic Behavior Study of Bacteria of Pseudomonas aeruginosa, Edwardsiella tarda and Enteropathogenic Escherichia coli by Capillary Electrophoresis with UV and Fluorescence Detection

Capillary electrophoresis approaches have been utilized for the study of bacteria under specific experimental conditions. The main objective within our research work was to study electrophoretic behaviors of Pseudomonas aeruginosa by means of capillary electrophoresis with UV and fluorescence detection. Edwardsiella tarda and Enteropathogenic escherichia coli were also included in the study. The results showed that proper pretreatment (vortexing or sonication) for each bacterial sample before injection was necessary to disperse the clusters of cells, which is helpful to observe the single peaks and better peak shape of bacteria during electrophoresis. Apart from this, it was found that ionic strength of buffer affected mobilities of Pseudomonas aeruginosa as a result of increasing of buffer concentration from 25 mM to 150 mM. Moreover, sharp and single peaks were still observed without significant increase of noise in the concentration range. Eventually, mixtures of bacteria were well separated under optimized separation conditions with UV and fluorescence detection. In the mean time, comparison of concentration sensitivities for Pseudomonas aeruginosa by UV and fluorescence detection was made. Blue light emitting diode induced fluorescence detection was found to be more sensitive (8.5-fold higher) than UV detection with home-made fluorescence detection system. Generally, proposed CE methods for the analysis of bacteria could be potentially valuable for the monitoring of bacteria contamination in real life.     

Short-chain reactive probes as tools to unravel the Pseudomonas aeruginosa quorum sensing regulon

In recent years, the world has seen a troubling increase in antibiotic resistance among bacterial pathogens. In order to provide alternative strategies to combat bacterial infections, it is crucial deepen our understanding into the mechanisms that pathogens use to thrive in complex environments. Most bacteria use sophisticated chemical communication systems to sense their population density and coordinate gene expression in a collective manner, a process that is termed “quorum sensing” (QS). The human pathogen Pseudomonas aeruginosa uses several small molecules to regulate QS, and one of them is N-butyryl-l-homoserine lactone (C₄-HSL). Using an activity-based protein profiling (ABPP) strategy, we designed biomimetic probes with a photoreactive group and a ‘click’ tag as an analytical handle. Using these probes, we have identified previously uncharacterized proteins that are part of the P. aeruginosa QS network, and we uncovered an additional role for this natural autoinducer in the virulence regulon of P. aeruginosa, through its interaction with PhzB1/2 that results in inhibition of pyocyanin production.

Short-chain reactive probes can be used as tools to shed new light on virulence mechanisms in bacterial pathogens.     

Dressings Loaded with Cyclodextrin–Hamamelitannin Complexes Increase Staphylococcus aureus Susceptibility Toward Antibiotics Both in Single as well as in Mixed Biofilm Communities

Bacteria reside within biofilms at the infection site, making them extremely difficult to eradicate with conventional wound care products. Bacteria use quorum sensing (QS) systems to regulate biofilm formation, and QS inhibitors (QSIs) have been proposed as promising antibiofilm agents. Despite this, few antimicrobial therapies that interfere with QS exist. Nontoxic hydroxypropyl‐β‐cyclodextrin‐functionalized cellulose gauzes releasing a burst of the antibiotic vancomycin and the QSI hamamelitannin are developed, followed by a sustained release of both. The gauzes affect QS and biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus in an in vitro model of chronic wound infection and can be considered as candidates to be used to prevent wound infection as well as treat infected wounds.

     

Synthesis,Antimicrobial, Anti-Virulence and Anticancer Evaluation of New 5(4H)-Oxazolone-Based Sulfonamides

Since the synthesis of prontosil the first prodrug shares their chemical moiety, sulfonamides exhibit diverse modes of actions to serve as antimicrobials, diuretics, antidiabetics, and other clinical applications. This inspiring chemical nucleus has promoted several research groups to investigate the synthesis of new members exploring new clinical applications. In this study, a novel series of 5(4H)-oxazolone-based-sulfonamides (OBS) 9a–k were synthesized, and their antibacterial and antifungal activities were evaluated against a wide range of Gram-positive and -negative bacteria and fungi. Most of the tested compounds exhibited promising antibacterial activity against both Gram-positive and -negative bacteria particularly OBS 9b and 9f. Meanwhile, compound 9h showed the most potent antifungal activity. Moreover, the OBS 9a, 9b, and 9f that inhibited the bacterial growth at the lowest concentrations were subjected to further evaluation for their anti-virulence activities against Pseudomonas aeruginosa and Staphylococcus aureus. Interestingly, the three tested compounds reduced the biofilm formation and diminished the production of virulence factors in both P. aeruginosa and S. aureus. Bacteria use a signaling system, quorum sensing (QS), to regulate their virulence. In this context, in silico study has been conducted to assess the ability of OBS to compete with the QS receptors. The tested OBS showed marked ability to bind and hinder QS receptors, indicating that anti-virulence activities of OBS could be due to blocking QS, the system that controls the bacterial virulence. Furthermore, anticancer activity has been further performed for such derivatives. The OBS compounds showed variable anti-tumor activities, specifically 9a, 9b, 9f and 9k, against different cancer lines. Conclusively, the OBS compounds can serve as antimicrobials, anti-virulence and anti-tumor agents.     

Microcalorimetric study on the growth and metabolism of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Microcalorimetry is an experimental technique which allows us to precisely measure the energy released as a consequence of any transformation process. All organisms produce heat as a consequence of metabolism. The rate of heat production is an adequate measurement of metabolic activity of organisms and their constituent parts, cells and sub-cellular levels. Microorganisms produce small amounts of heat, in the order of 1–3 pW per cell. Despite the low quantity of heat produced by bacteria, their exponential replication in culture medium allows their detection using microcalorimetry. This study is a microcalorimetric study of the growth and metabolism of the bacterium Pseudomonas aeruginosa, using the heat liberated as a consequence of bacterial metabolism. With this aim, we used a Calvet microcalorimeter, inside which two Teflon screw-capped stainless steel cells were located (sample and reference). Experiments were carried out at final concentrations of 10⁶, 10⁵, 10³ and 10 CFU/mL, and a constant temperature of 309.65 K was maintained within the microcalorimeter. Recording the difference in calorific potential over time we obtained P. aeruginosa’s growth curves. The shape of these curves is characteristic and has a single phase. Thus, the heat flow curves were mathematically studied to calculate the growth constant and generation time of this bacterium.     

Role of the Quorum Sensing Mechanism in the Response of Pseudomonas aeruginosa to Lethal and Sublethal UVA Irradiation

The role of quorum sensing (QS) in the response of Pseudomonas aeruginosa to UVA radiation was investigated in the PAO1 strain and derivatives defective in the synthesis of the QS signals 3OC12-HSL (lasI strain), C4-HSL (rhlI strain) or both (lasI rhlI strain). Cell viability measurements demonstrated that the double mutant was significantly more sensitive to UVA than single mutants, which in turn showed reduced cell survival with regard to the PAO1 strain. Irradiation under nitrogen atmosphere and chemiluminescence measurements indicated the oxidative nature of the UVA-induced damage. The activity of the antioxidant enzymes catalase and superoxide dismutase was assayed in these strains before and after irradiation, and a strong correlation between catalase levels and UVA sensitivity was observed. When a sublethal UVA dose was applied to PAO1, a growth delay was observed and this mechanism depended on the rhl system. Moreover, low doses of UVA irradiation triplicated the level of C4-HSL in log PAO1 cells. It is concluded that QS is fundamental in the defense against the toxic effects of UVA in P. aeruginosa. The induction of the QS system by UVA independently of cell density could function as an adaptative strategy to withstand this hostile environmental condition.     

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.     

Quorum sensing regulates electric current generation of Pseudomonas aeruginosa PA14 in bioelectrochemical systems

Here, we show that quorum sensing (QS) modulates the current generation of the anode-respiring bacterium Pseudomonas aeruginosa because it controls the production of phenazines, which mediate the electron transfer to the anode. The current generation by a wildtype (WT) strain P. aeruginosa PA14 and the GacS/GacA protein-regulatory mutant retS was investigated under different environmental conditions. The retS mutant generated significantly higher current (45-fold) than the WT under anaerobic conditions. Anaerobic current generation by the WT was 28-fold higher with extraneously supplied lactones (a QS-signaling molecule). Compared to anaerobic conditions, the WT with some oxygen (microaerobic conditions) exhibited enhanced phenazine production (39-fold) and current levels (48-fold). Iron-rich medium and microaerobic conditions had a negative impact on current generation by retS. All these results were directly linked to QS activity in P. aeruginosa, thus, demonstrating the importance of this bacterial communication system for current generation in BESs. We also show that BESs represent a new tool for real-time investigation of phenazine-related QS activity.