Bacterial Autoinducers Research Articles

Microbead-Encapsulated Luminescent Bioreporter Screening of P. aeruginosa via Its Secreted Quorum-Sensing Molecules.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that remains a prevalent clinical and environmental challenge. Quorum-sensing (QS) molecules are effective biomarkers in pinpointing the presence of P. aeruginosa. This study aimed to develop a convenient-to-use, whole-cell biosensor using P. aeruginosa reporters individually encapsulated within alginate-poly-L-lysine (alginate-PLL) microbeads to specifically detect the presence of bacterial autoinducers. The PLL-reinforced microbeads were prepared using a two-step method involving ionic cross-linking and subsequent coating with thin layers of PLL. The alginate-PLL beads showed good stability in the presence of a known cation scavenger (sodium citrate), which typically limits the widespread applications of calcium alginate. In media containing synthetic autoinducers-such as N-(3-oxo dodecanoyl) homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL), or the cell-free supernatants of planktonic or the flow-cell biofilm effluent of wild P. aeruginosa (PAO1)-the encapsulated bacteria enabled a dose-dependent detection of the presence of these QS molecules. The prepared bioreporter beads remained stable during prolonged storage at 4 and -80 °C and were ready for on-the-spot sensing without the need for recovery. The proof-of-concept, optical fiber-based, and whole-cell biosensor developed here demonstrates the practicality of the encapsulated bioreporter for bacterial detection based on specific QS molecules.

Ibuprofen involves with the reduced expression of pelD and pelF in pathogenic Pseudomonas aeruginosa strains.

Biofilm formation is an important factor in disease development by Pseudomonas aeruginosa. Similar to many bacterial species, biofilm formation in P. aeruginosa is regulated by the bacterial quorum sensing system. The pel genes are responsible for the synthesis of a glucose-rich polysaccharide that is associated with biofilm initiation and maturation. The antibiofilm potential of ibuprofen has been reported; however, the effect of the drug on the expression of the genes involved with biofilm formation has rarely been described. In this work, the effect of ibuprofen on the biofilm formation and expression of pelD and pelF genes among pathogenic P. aeruginosa strains was investigated. Multiple drug-resistant P. aeruginosa strains were treated with ibuprofen at ½ MIC concentration and their biofilm formation and expression of pelD and pelF genes was determined using the crystal violet and real-time PCR assays, respectively. The results showed that the ibuprofen at 1024µg/mL significantly reduced biofilm formation of P. aeruginosa strains by 52-77%, compared with the controls. In addition, treating the bacteria with ibuprofen decreased the expression of pelD and pelF genes to 0.56 and 0.69 folds, respectively. We hypothesized that the attenuation of the pel genes could be associated with the reduction of bacterial QS autoinducers, which in turn reduced cellular c-di-GMP level. This work suggests that ibuprofen is a potent antibiofilm drug that could be used to enhance bacterial susceptibility to antimicrobials through the inhibition of biofilm formation.

Quorum-sensing signaling by chironomid egg masses' microbiota, affects haemagglutinin/protease (HAP) production by Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, is commonly isolated, along with other bacterial species, from chironomid insects (Diptera: Chironomidae). Nevertheless, its prevalence in the chironomid egg masses' microbiota is less than 0.5%. V. cholerae secretes haemagglutinin/protease (HAP) that degrades the gelatinous matrix of chironomid egg masses and prevents hatching. Quorum sensing (QS) activates HAP production in response to accumulation of bacterial autoinducers (AIs). Our aim was to define the impact of chironomid microbiota on HAP production by V.cholerae. To study QS signaling, we used V.cholerae bioluminescence reporter strains (QS-proficient O1 El-Tor wild-type and QS-deficient mutants) and different bacterial species that we isolated from chironomid egg masses. These egg mass isolates, as well as a synthetic AI-2, caused an enhancement in lux expression by a V.cholerae QS-deficient mutant. The addition of the egg mass bacterial isolate supernatant to the QS-deficient mutant also enhanced HAP production and egg mass degradation activities. Moreover, the V.cholerae wild-type strain was able to proliferate using egg masses as their sole carbon source, while the QS-deficient was not. The results demonstrate that members of the chironomid bacterial consortium produce external chemical cues that, like AI-2, induce expression of the hapA gene in V.cholerae. Understanding the interactions between V.cholerae and the insects' microbiota may help uncover the interactions between this pathogen and the human gut microbiota.

Inter-kingdom signaling between gut microbiota and their host.

The crosstalk between prokaryotic bacteria and eukaryotic gut epithelial cells has opened a new field for research. Quorum sensing system (QS) molecules employed by gut microbiota may play an essential role in host-microbial symbioses of the gut. Recent studies on the gut microbiome will unveil evolved mechanisms of the host to affect bacterial QS and shape bacterial composition. Bacterial autoinducers (AIs) could talk to the host's gut by eliciting proinflammatory effects and modulating the activities of T lymphocyte, macrophage, dendritic cells, and neutrophils. In addition, the gut mucosa could interfere with bacterial AIs by degrading them or secreting AI mimics. Moreover, bacterial AIs and gut hormones epinephrine and noradrenaline may be interchangeable in the crosstalk between the microbiota and human gut. Therefore, inter-kingdom signaling between gut microbiota and host may provide a novel target in the management of gut microbiota-related conditions or diseases in the future.

Synthetic nanoparticles for selective hydrolysis of bacterial autoinducers in quorum sensing

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

A Versatile Redox Responsive Nanoferrogels Based Sensor for Metabolics Analytics

Developed is an inexpensive label free redox responsive nanoferrogel molecular receptor integrated in a chemoresisistive sensor for rapid detection of Reactive Oxygen Species (ROS) such as H2O2 and H2O2 deriving metabolites, species are of interest in myriad applications including clinical, environmental, food, and plant physiology. Inexpensively fashioned by contact printing of redox responsive poly(N-isopropylacrylamide hydrogels herewith called ‘nanoferrogel’ on a conductive nanocellulose paper aerogel, the sensor is designed for specimen surface attachment as wearables, non-invasive transdermal sensors, and can be configured as microneedle sensor for in vivo analysis. Operating via ROS oxidation of the nanoferrogel trapped in the conductive aerogel film, the oxidized nanoferrogel volume change results in an overall change in conductivity, linearly proportional to ROS/ROS deriving metabolite concentration. In the presentation, the responsivity of the nanoferrogel to different ROS species such as H2O2, oxylipin, antioxidants will be demonstrated. The nanoferrogel based H2O2 sensor figures of merit, featuring, rapid (≤ 1min) H2O2 response, high selectivity, low limits of detection (≤ 0.01 µM),and broad linear dynamic range of (≤ 0.03 µM to ≥ 1.5M) will be highlighted. The nanoferrogel is perfectly recyclable, by electrochemical regeneration, affording ≥ 15 multiple usable cycles. Also showcased will be the adaptability of nanoferrogel for different sensor design configurations (e.g. micro/nanoelectrode systems), with some preliminary data attesting to the sensors utility for fundamental studies and detection of efflux of endogenous and exogenous H2O2 in single cells and other redox biomolecules, (e.g. glucose, choline). Preliminarily the sensor can be used for detection of bacterial (model used in our study Escherichia coli) autoinducers and metabolites, of importance in bacterial communication-quorum sensing. This would be a useful platform for understanding bacterial biofilm formation. Further, the nanoferrogel sensor fashioned for utility as a wearable device for detection of redox potential and inherent sweat metabolites will be demonstrated.

The Dynamic Interactions between Salmonella and the Microbiota, within the Challenging Niche of the Gastrointestinal Tract

Understanding how Salmonella species establish successful infections remains a foremost research priority. This gastrointestinal pathogen not only faces the hostile defenses of the host's immune system, but also faces fierce competition from the large and diverse community of microbiota for space and nutrients. Salmonella have solved these challenges ingeniously. To jump-start growth, Salmonella steal hydrogen produced by the gastrointestinal microbiota. Type 3 effector proteins are subsequently secreted by Salmonella to trigger potent inflammatory responses, which generate the alternative terminal electron acceptors tetrathionate and nitrate. Salmonella exclusively utilize these electron acceptors for anaerobic respiration, permitting metabolic access to abundant substrates such as ethanolamine to power growth blooms. Chemotaxis and flagella-mediated motility enable the identification of nutritionally beneficial niches. The resulting growth blooms also promote horizontal gene transfer amongst the resident microbes. Within the gastrointestinal tract there are opportunities for chemical signaling between host cells, the microbiota, and Salmonella. Host produced catecholamines and bacterial autoinducers form components of this chemical dialogue leading to dynamic interactions. Thus, Salmonella have developed remarkable strategies to initially shield against host defenses and to transiently compete against the intestinal microbiota leading to successful infections. However, the immunocompetent host is subsequently able to reestablish control and clear the infection.

Phosphate concentration alters the effective bacterial quorum in the symbiosis of Medicago truncatula-Sinorhizobium meliloti

The symbiosis of Medicago truncatula-Sinorhizobium meliloti is affected by phosphate (P) deficiency in the environment. Quorum sensing (QS) is a regulatory pathway in S. meliloti that controls various functions of free-living and symbiotic bacteria in response to phosphate availability and regulation is mediated by a periplasmic protein PstS, and also bacterial density. The quorum sensing pathway of S. meliloti, involves three genes named sinI, sinR and expR and also some bacterial auto-inducers such as N-acyl homoserine lactones (AHLs). In the current study, the expression of the different genes of quorum sensing and pstS were evaluated under 0.1, 0.5 and 2 mM P. The qRT-PCR results showed an increased expression of pstS and also the quorum sensing genes sinI and sinR but not expR, following phosphate starvation. Indeed, the enhanced level of sinR induces the expression of sinI that is responsible for the N-acyl homoserine lactones (AHL) production in S. meliloti. The different response of expR may be due to its negative control on sinR expression. In the symbiosis of M. truncatula-S. meliloti, it was shown that the concentration of phosphate in the medium alters the effective inoculating bacterial quorum (density). By increasing the phosphate concentration in the medium from 0.1 to 0.5 and 2 mM, considering the optimal plant growth and pink nodule (nitrogen-fixing) formation, the effective inoculating bacterial densities were 105, 107 and 109 CFU ml−1, respectively. Therefore, low phosphate concentrations can compensate for a low bacterial density by inducing the quorum sensing pathway and establishing a symbiosis. Conversely, bacterial density plays the main role in the formation of symbiosis at high phosphate concentrations.

Role of Pseudomonas aeruginosa quorum sensing (QS) molecules on the viability and cytokine profile of human mesenchymal stem cells

Pseudomonas aeruginosa infections represent one of the major threats for injured or transplanted lungs and for their healing. Considering that the mesenchymal stem cells (MSCs) are a major tool for the regenerative medicine, including therapy of lung damaging diseases, the aim of this paper was to investigate the effects of P. aeruginosa quorum sensing signaling molecules (QSSMs) on human MSCs death signaling pathways and cytokine profile. Our data revealed that N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), N-butanoyl-L-homoserine lactone (C4-HSL), 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), and its precursor, 2-heptyl-4-quinolone (HHQ), significantly impact on several core signaling mechanisms of MSCs in a specific and time-dependent manner. Even if all tested autoinducers interfered with the MSCs apoptotic genes expression, only OdDHL and HHQ significantly promoted MSCs apoptosis, by 14- and 23-fold respectively, this aspect being confirmed by the flow cytometry assay. The tested QSSMs induced a heterogeneous cytokine profile of the treated MSCs. The level of IL-1β was increased by OdDHL, IL-8 production was stimulated by all tested autoinducers, IL-6 was modulated mostly by PQS and IL-10 by HHQ. The significant influence of the purified bacterial autoinducers on the MSCs signaling pathways may suggest that the accumulation of these mediators could interfere with the normal function of these cells in the human body, and eventually, impair or abolish the success of the stem cells therapy during P. aeruginosa infections.

Characterization of an Autoinducer of Penicillin Biosynthesis in Penicillium chrysogenum

Filamentous fungi produce an impressive variety of secondary metabolites; many of them have important biological activities. The biosynthesis of these secondary metabolites is frequently induced by plant-derived external elicitors and appears to also be regulated by internal inducers, which may work in a way similar to that of bacterial autoinducers. The biosynthesis of penicillin in Penicillium chrysogenum is an excellent model for studying the molecular mechanisms of control of gene expression due to a good knowledge of the biochemistry and molecular genetics of β-lactam antibiotics and to the availability of its genome sequence and proteome. In this work, we first developed a plate bioassay that allows direct testing of inducers of penicillin biosynthesis using single colonies of P. chrysogenum. Using this bioassay, we have found an inducer substance in the conditioned culture broths of P. chrysogenum and Acremonium chrysogenum. No inducing effect was exerted by γ-butyrolactones, jasmonic acid, or the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine. The conditioned broth induced penicillin biosynthesis and transcription of the pcbAB, pcbC, and penDE genes when added at inoculation time, but its effect was smaller if added at 12 h and it had no effect when added at 24 h, as shown by Northern analysis and lacZ reporter studies. The inducer molecule was purified and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as 1,3-diaminopropane. Addition of pure 1,3-diaminopropane stimulated the production of penicillin by about 100% compared to results for the control cultures. Genes for the biosynthesis of 1,3-diaminopropane have been identified in the P. chrysogenum genome.

A complex relationship: the interaction among symbiotic microbes, invading pathogens, and their mammalian host.

Symbiosis between microbes and their mammalian host is vital to maintaining homeostasis. Symbiotic microbes within the gastrointestinal tract provide an array of benefits to the host, including promotion of host immunity. A coordinated effort of the host and symbiotic microbes deters the colonization and survival of many invading pathogens. However, pathogens have devised strategies to overcome these mechanisms. Furthermore, some pathogens can hijack host hormones and bacterial autoinducers to induce virulence traits. Intra- and inter-species (bacteria: bacteria) and inter-kingdom (bacteria: host) communication orchestrates the complex relationship among symbiotic microbes, invading pathogens, and their mammalian host. Insight into this communication will provide a foundation for the development of targeted antimicrobial therapies.

Biological Nanofactories Target and Activate Epithelial Cell Surfaces for Modulating Bacterial Quorum Sensing and Interspecies Signaling

In order to control the behavior of bacteria present at the surface of human epithelial cells, we have created a biological "nanofactory" construct that "coats" the epithelial cells and "activates" the surface to produce the bacterial quorum sensing signaling molecule, autoinducer-2 (AI-2). Specifically, we demonstrate directed modulation of signaling among Escherichia coli cells grown over the surface of human epithelial (Caco-2) cells through site-directed attachment of biological nanofactories. These "factories" comprise a fusion protein expressed and purified from E. coli containing two AI-2 bacterial synthases (Pfs and LuxS), a protein G IgG binding domain, and affinity ligands for purification. The final factory is fabricated ex vivo by incubating with an anti-CD26 antibody that binds the fusion protein and specifically targets the CD26 dipeptidyl peptidase found on the outer surface of Caco-2 cells. This is the first report of the intentional "in vitro" synthesis of bacterial autoinducers at the surface of epithelial cells for the redirection of quorum sensing behaviors of bacteria. We envision tools such as this will be useful for interrogating, interpreting, and disrupting signaling events associated with the microbiome localized in human intestine and other environments.

QS-type bacterial signal molecules of nonpeptide origin

This review classifies and analyzes the literature data on bacterial autoinducers (AI), the signal molecules produced and secreted by bacterial cells and responsible for intercellular communication (quorum sensing, QS). The most important families of nonpeptide AI are discussed, including N-acyl homoserine lactones, derivatives of 2-methyl-2,3,4,5-tetrahydroxy tetrahydrofuran, indole and quinoline derivatives, and adrenalinerelated compounds. The data is provided on the intracellular and membrane receptors specifically binding to AI, as well as on the effector systems that are activated by AI and mediate their regulatory effects. The possible role of some vertebrate hormones (adrenergic agonists, serotonin, etc.) as AI and their effect on bacterial activity are discussed. The data are presented suggesting a high efficiency of AI-based antibacterial preparations, which selectively disrupt the bacterial information network and thus suppress bacterial infections.

Hormone fatty acid modifications: Gram negative bacteria and vertebrates demonstrate common structure and function

Bacteria are known to regulate diverse physiological processes through a mechanism called quorum sensing (QS). Prokaryotes communicate by extracellular signalling compounds, i.e. autoinducers (acyl homoserine lactone, AHL of Gram negative bacteria) or pheromones (post-translationally modified peptides of Gram positive bacteria), which activate genetic pathways when they reach a sufficient concentration (QS). A large number of Gram-negative quorum-sensing systems studied so far utilize N-acyl homoserine lactones as signal molecules. In vertebrates small synthetic molecules called growth hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. GH release is stimulated by hypothalamic GH-releasing hormone (GHRH) and ghrelin (endogenous ligand of the GHS-receptor, GHS-R). Ghrelin is a 28-amino acid peptide, in which the serine-3 (Ser3) is n-octanoylated, and this modification is essential for ghrelin's activity. Ghrelin is the first known case of a peptide hormone modified by a fatty acid. The major active form of ghrelin is a 28-amino acid peptide with octanoylated Ser3; one of the more represented bacterial autoinducers is the N-Octanoyl-DL-homoserine lactone (C8-HL) molecule. The authors hypothesize that Gram-negative bacteria and vertebrates have a functional similarity in the search of food and an important structural homology of AHL and ghrelin for the highly conserved Serine-acylated motive in both molecules. Our suggestions could help one to understand the convergent origin and the biologic meaning of the Serine-acylated group in these organisms, a biologic meaning very important due to the high conservation in two kingdoms which are so different.

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.

Quorum sensing in bacteria.

Quorum sensing is the regulation of gene expression in response to fluctuations in cell-population density. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. Gram-positive and Gram-negative bacteria use quorum sensing communication circuits to regulate a diverse array of physiological activities. These processes include symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation. In general, Gram-negative bacteria use acylated homoserine lactones as autoinducers, and Gram-positive bacteria use processed oligo-peptides to communicate. Recent advances in the field indicate that cell-cell communication via autoinducers occurs both within and between bacterial species. Furthermore, there is mounting data suggesting that bacterial autoinducers elicit specific responses from host organisms. Although the nature of the chemical signals, the signal relay mechanisms, and the target genes controlled by bacterial quorum sensing systems differ, in every case the ability to communicate with one another allows bacteria to coordinate the gene expression, and therefore the behavior, of the entire community. Presumably, this process bestows upon bacteria some of the qualities of higher organisms. The evolution of quorum sensing systems in bacteria could, therefore, have been one of the early steps in the development of multicellularity.

The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule.

Many bacteria control gene expression in response to cell population density, and this phenomenon is called quorum sensing. In Gram-negative bacteria, quorum sensing typically involves the production, release and detection of acylated homoserine lactone signalling molecules called autoinducers. Vibrio harveyi, a Gram-negative bioluminescent marine bacterium, regulates light production in response to two distinct autoinducers (AI-1 and AI-2). AI-1 is a homoserine lactone. The structure of AI-2 is not known. We have suggested previously that V. harveyi uses AI-1 for intraspecies communication and AI-2 for interspecies communication. Consistent with this idea, we have shown that many species of Gram-negative and Gram-positive bacteria produce AI-2 and, in every case, production of AI-2 is dependent on the function encoded by the luxS gene. We show here that LuxS is the AI-2 synthase and that AI-2 is produced from S-adenosylmethionine in three enzymatic steps. The substrate for LuxS is S-ribosylhomocysteine, which is cleaved to form two products, one of which is homocysteine, and the other is AI-2. In this report, we also provide evidence that the biosynthetic pathway and biochemical intermediates in AI-2 biosynthesis are identical in Escherichia coli, Salmonella typhimurium, V. harveyi, Vibrio cholerae and Enterococcus faecalis. This result suggests that, unlike quorum sensing via the family of related homoserine lactone autoinducers, AI-2 is a unique, 'universal' signal that could be used by a variety of bacteria for communication among and between species.

ChemInform Abstract: Chemical Synthesis of Bacterial Autoinducers and Analogues

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: A Novel Convenient Route to the Naturally Occurring 3‐Oxoacyl‐L‐ homoserine Lactones and Related Bacterial Autoinducers.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

A novel convenient route to the naturally occurring 3-oxoacyl-L-homoserinelactones and related bacterial autoinducers

The naturally occurring 3-oxohexanoyl-L-homoserinelactone ( 1a), a bacterial autoinducer, has been prepared in 47 % overall yield by condensing stable 3-oxohexanoic acid ( 2), prepared by hydrolysis from the corresponding ester (3), with L-homoserinelactone using hydroxybenzotriazole (HOBT) and dicyclohexylcarbodiimide (DCC) in non-aqueous media.