Presence Of Acyl-homoserine Lactones Research Articles

High-Throughput Virtual Screening for a New Class of Antagonist Targeting LasR of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic human pathogen, causes fatal effects in patients with cystic fibrosis and immunocompromised individuals and leads to around 1000 deaths annually. The quorum sensing mechanism of P. aeruginosa plays a major role in promoting biofilm formation and expression of virulent genes. Hence, quorum sensing inhibition is a promising novel approach to treat these bacterial infections as these organisms show a wide range of antibiotic resistance. Among the interconnected quorum sensing network of P. aeruginosa, targeting the las system is of increased interest as its principal receptor protein LasR is the earliest activated gene. It is also shown to be involved in the regulation of other virulence-associated genes. In this study, we have applied high-throughput virtual screening, an in silico computational method to identify a new class of LasR inhibitors that could serve as potent antagonists to treat P. aeruginosa-associated infections. Three-tire structure-based virtual screening was performed on the Schrödinger small molecule database, which resulted in 12 top hit compounds with docking scores lesser than −11.0 kcal/mol. Three of these best-scored compounds CACPD2011a-0001928786 (C1), CACPD2011a-0001927437 (C2), and CACPD2011a-0000896051 (C3) were further analyzed. The binding free energies of these compounds in complex with the target protein LasR (3IX4) were evaluated, and the pharmacokinetic properties were determined. The stability of the docked complexes was assessed by running a molecular dynamics simulation for 100 ns. Molecular dynamics simulation analysis revealed that all three compounds were found to be in stable contact with the protein over the entire simulation period. The antagonistic effect of these compounds was validated using the LasR reporter gene assay in the presence of acyl homoserine lactone. Significant reduction in the β-galactosidase enzyme activity was achieved at 100 nM concentration for all three compounds pursued. Hence, the present study provides strong evidence that these three compounds could serve as quorum sensing inhibitors of P. aeruginosa LasR protein and can be a probable candidate to treat Pseudomonas-associated infections.

Accelerating the start-up of the cathodic biofilm by adding acyl-homoserine lactone signaling molecules

Electroactive biofilms (EABs) are essential for bioelectrochemical systems, however, the formation of cathodic EABs is more time-consuming than anodic EABs. This study aims to evaluate whether acyl-homoserine lactones (AHLs) could facilitate the start-up of cathodic Geobacter soli EABs. With AHL addition, the biomass, cell viability, and extracellular polymeric substance (EPS) abundance of cathode-associated G. soli EABs were increased. Likewise, redox activities of EPS and outermost proteins in the cathodic EABs were enhanced in the presence of AHLs, which consequently led to better start-up performance of biofilms. Compared to the control without AHLs, start-up lag periods were reduced by approximately 50%, electron uptakes were enhanced by 1.3–2.0 times, and denitrification rates were more than doubled with AHL addition in the start-up cycle, which were comparable to those of mature G. soli cathodic EABs. These findings open up an opportunity for accelerating the start-up of cathodic biofilms via AHLs.

Transcriptome Profiling Reveals the EanI/R Quorum Sensing Regulon in Pantoea Ananatis LMG 2665T.

Pantoea ananatis LMG 2665T synthesizes and utilizes acyl homoserine lactones (AHLs) for signalling. The complete set of genes regulated by the EanI/R quorum sensing (QS) system in this strain is still not fully known. In this study, RNA-sequencing (RNA-seq) was used to identify the EanI/R regulon in LMG 2665T. Pairwise comparisons of LMG 2665T in the absence of AHLs (Optical density (OD)600 = 0.2) and in the presence of AHLs (OD600 = 0.5) were performed. Additionally, pairwise comparisons of LMG 2665T and its QS mutant at OD600 = 0.5 were undertaken. In total, 608 genes were differentially expressed between LMG 2665T at OD600 = 0.5 versus the same strain at OD600 = 0.2 and 701 genes were differentially expressed between LMG 2665T versus its QS mutant at OD600 = 0.5. A total of 196 genes were commonly differentially expressed between the two approaches. These constituted approximately 4.5% of the whole transcriptome under the experimental conditions used in this study. The RNA-seq data was validated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Genes found to be regulated by EanI/R QS were those coding for redox sensing, metabolism, flagella formation, flagella dependent motility, cell adhesion, biofilm formation, regulators, transport, chemotaxis, methyl accepting proteins, membrane proteins, cell wall synthesis, stress response and a large number of hypothetical proteins. The results of this study give insight into the genes that are regulated by the EanI/R system in LMG 2665T. Functional characterization of the QS regulated genes in LMG 2665T could assist in the formulation of control strategies for this plant pathogen.

Changes in the Salmonella enterica Enteritidis phenotypes in presence of acyl homoserine lactone quorum sensing signals.

Quorum sensing is used by bacteria to coordinate gene expression in response to population density and involves the production, detection and response to extracellular signaling molecules known as autoinducers (AIs). Salmonella does not synthesize the AI-1, acyl homoserine lactone (AHL) common to gram-negative bacteria; however, it has a receptor for AI-1, the SdiA protein. The effect of SdiA in modulating phenotypes of Salmonella has not been elucidated. In this report, we provide evidence that the AIs-1 affect Salmonella enterica serovar Enteritidis behavior by enhancing the biofilm formation and expression of virulence genes under anaerobic conditions. Biofilm formation by Salmonella was detected by the crystal violet method and by scanning electron microscopy. The presence of AHLs, particularly C12-HSL, increased biofilm formation and promoted expression of biofilm formation genes (lpfA, fimF, fliF, glgC) and virulence genes (hilA, invA, invF). Our results demonstrated that AHLs produced by other organisms played an important role in virulence phenotypes of Salmonella Enteritidis.

Acyl-homoserine lactone recognition and response hindering the quorum-sensing regulator EsaR.

During quorum sensing in the plant pathogen Pantoea stewartii subsp. stewartii, EsaI, an acyl-homoserine lactone (AHL) synthase, and the transcription factor EsaR coordinately control capsular polysaccharide production. The capsule is expressed only at high cell density when AHL levels are high, leading to inactivation of EsaR. In lieu of detailed structural information, the precise mechanism whereby EsaR recognizes AHL and is hindered by it, in a response opposite to that of most other LuxR homologues, remains unresolved. Hence, a random mutagenesis genetic approach was designed to isolate EsaR* variants that are immune to the effects of AHL. Error-prone PCR was used to generate the desired mutants, which were subsequently screened for their ability to repress transcription in the presence of AHL. Following sequencing, site-directed mutagenesis was used to generate all possible mutations of interest as single, rather than multiple amino acid substitutions. Eight individual amino acids playing a critical role in the AHL-insensitive phenotype have been identified. The ability of EsaR* variants to bind AHL and the effect of individual substitutions on the overall conformation of the protein were examined through in vitro assays. Six EsaR* variants had a decreased ability to bind AHL. Fluorescence anisotropy was used to examine the relative DNA binding affinity of the final two EsaR* variants, which retained some AHL binding capability but remained unresponsive to it, perhaps due to an inability of the N-terminal domain to transduce information to the C-terminal domain.

The Acyl-Homoserine Lactone Synthase YenI from Yersinia enterocolitica Modulates Virulence Gene Expression in Enterohemorrhagic Escherichia coli O157:H7

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes the rectoanal junction (RAJ) in cattle, its natural reservoir. Colonization at the RAJ poses a serious risk for fecal shedding and contamination of the environment. We previously demonstrated that EHEC senses acyl-homoserine lactones (AHLs) produced by the microbiota in the rumen to activate the gad acid resistance genes necessary for survival through the acidic stomachs in cattle and to repress the locus of enterocyte effacement (LEE) genes important for colonization of the RAJ, but unnecessary in the rumen. Devoid of AHLs, the RAJ is the prominent site of colonization of EHEC in cattle. To determine if the presence of AHLs in the RAJ could repress colonization at this site, we engineered EHEC to express the Yersinia enterocolitica AHL synthase gene yenI, which constitutively produces AHLs, to mimic a constant exposure of AHLs in the environment. The yenI(+) EHEC produces oxo-C6-homoserine lactone (oxo-C6-HSL) and had a significant reduction in LEE expression, effector protein secretion, and attaching and effacing (A/E) lesion formation in vitro compared to the wild type (WT). The yenI(+) EHEC also activated expression of the gad genes. To assess whether AHL production, which decreases LEE expression, would decrease RAJ colonization by EHEC, cattle were challenged at the RAJ with WT or yenI(+) EHEC. Although the yenI(+) EHEC colonized the RAJ with efficiency equal to that of the WT, there was a trend for the cattle to shed the WT strain longer than the yenI(+) EHEC.

Signaling Mechanisms and Physical Structure of Biofilm Growth in Synechocystis sp. Strain PCC 6803

Chemical signaling has been found in the past to play a central role in bacterial biofilm formation, and may be important for the cyanobacterium Synechocystis sp. Strain PCC 6803. We investigate the cell‐cell communication system of this species and whether it involves acyl homoserine lactone (AHL) signaling molecules. We use the biosensor, Chromobacterium violaceum CV026, which produces the violacein pigment in the presence of AHLs (McClean, et al., 1997). This biosensor was found to be a sensitive and quantitative method of detecting a variety of AHL chain lengths. Optimal growth temperature is 29°C and growth is not affected at low concentrations of acid, which is required for AHL stability. The physical structure of the biofilms of a super motile cyanobacterial mutant is also being analyzed using confocal microscopy. The biofilms begin as pillars 20–30um tall, grow to 50um deep and spread across the surface. The motile cyanobacterial cells exhibit unique biofilm formation and structure, and we are determining whether the process involves AHL signaling. This research is supported by the Wellesley College Biological Sciences Department and a Jerome A. Schiff Fellowship.

Characterization of a quorum sensing device for synthetic biology design: Experimental and modeling validation

Modeling of biological parts is of crucial importance as it enables the in silico study of synthetic biological systems prior to the actual construction of genetic circuits, which can be time consuming and costly. Because standard biological parts are utilized to build the synthetic systems, it is important that each of these standard parts is well characterized and has a corresponding mathematical model that could simulate the characteristics of the part. These models could be used in computer aided design (CAD) tools during the design stage to facilitate the building of the model of biological systems. This paper describes the development of a mathematical model that is able to simulate both the dynamic and static performance of a biological device created using standard parts. We modeled an example quorum sensing device that produces green fluorescent protein (GFP) as reporter in the presence of Acyl Homoserine Lactone (AHL). The parameters of the model were estimated using experimental results. The simulation results show that the model was able to simulate behavior similar to experimental results. Since it is important that these models and the content in the models can be searchable and readable by machines, standard SBML (system biology markup language) format was used to store the models. All parts and reactions are fully annotated to enable easy searching, and the models follow the Minimum Information Requested In the Annotation of Models (MIRIAM) compliance as well as the Minimum Information About a Simulation Experiment (MIASE).

Probing the Impact of Ligand Binding on the Acyl-Homoserine Lactone-Hindered Transcription Factor EsaR of Pantoea stewartii subsp. stewartii

The quorum-sensing regulator EsaR from Pantoea stewartii subsp. stewartii is a LuxR homologue that is inactivated by acyl-homoserine lactone (AHL). In the corn pathogen P. stewartii, production of exopolysaccharide (EPS) is repressed by EsaR at low cell densities. However, at high cell densities when high concentrations of its cognate AHL signal are present, EsaR is inactivated and derepression of EPS production occurs. Thus, EsaR responds to AHL in a manner opposite to that of most LuxR family members. Depending on the position of its binding site within target promoters, EsaR serves as either a repressor or activator in the absence rather than in the presence of its AHL ligand. The effect of AHL on LuxR homologues has been difficult to study in vitro because AHL is required for purification and stability. EsaR, however, can be purified without AHL enabling an in vitro analysis of the response of the protein to ligand. Western immunoblots and pulse-chase experiments demonstrated that EsaR is stable in vivo in the absence or presence of AHL. Limited in vitro proteolytic digestions of a biologically active His-MBP tagged version of EsaR highlighted intradomain and interdomain conformational changes that occur in the protein in response to AHL. Gel filtration chromatography of the full-length fusion protein and cross-linking of the N-terminal domain both suggest that this conformational change does not impact the multimeric state of the protein. These findings provide greater insight into the diverse mechanisms for AHL responsiveness found within the LuxR family.

Biofilm formation and acyl homoserine lactone production in Hafnia alvei isolated from raw milk.

The objective of this study was to detect the presence of acyl homoserine lactones (AHLs), signal molecules of the quorum sensing system in biofilm formed by Hafnia alvei strains. It also evaluated the effect of synthetic quorum sensing inhibitors in biofilm formation. AHLs were assayed using well diffusion techniques, thin layer chromatography (TLC) and detection directly in biofilm with biomonitors. The extracts obtained from planktonic and sessile cell of H. alvei induced at least two of three monitor strains evaluated. The presence of AHLs with up to six carbon atoms was confirmed by TLC. Biofilm formation by H. alvei was inhibited by furanone, as demonstrated by 96-well assay of crystal violet in microtitre plates and by scanning electron microscopy. The H. alvei 071 hall mutant was deficient in biofilm formation. All these results showed that the quorum sensing system is probably involved in the regulation of biofilm formation by H. alvei.

SdiA Bridges Chemical Signaling between Salmonella enterica Serovar Typhimurium and Yersinia enterocolitica in Mice

The role of SdiA quorum-sensing-mediated signaling in Salmonella enterica serovar Typhimurium has been a matter of debate for many years. Although Salmonella harbors SdiA, it does not produce an acyl homoserine lactone (AHL) signal (8, 13). Many of the phenotypes associated with SdiA have been shown using sdiA cloned in multicopy plasmids (14). The precise role of SdiA in quorum sensing was elusive for several years until Michael et al. (8) reported that SdiA is not sensing an autoinducer produced by Salmonella itself but rather AHLs produced by other bacterial species. These authors also reported that SdiA-dependent phenotypes could be observed only in the presence of AHLs. These results are consistent with reports that most LuxR-type proteins use the AHL autoinducer as a “cofactor” to allow proper protein folding and that in the absence of this signal, the protein is targeted to degradation (10, 16, 17). The nuclear magnetic resonance structure of the SdiA protein has been solved and shows that SdiA uses AHL as a folding switch and requires binding of this signal to allow proper protein folding (15). SdiA regulates a few genes in Salmonella, including one gene involved in resistance to human complement, rck, and a predicted type III secreted effector, srgE (2). However, mutation of the sdiA gene had no effect on virulence of Salmonella in mouse, chicken, or bovine models of disease (1). This lack of phenotypes “in vivo” could be a result of Salmonella not encountering AHLs in these infection models. The first clue that this might be the case occurred when it was found that turtles colonized by Aeromonas hydrophila (a bacterium that produces AHLs) activated SdiA function in these animals (12). Now Dyszel et al. (5) have shown that in mice coinfected with Salmonella serovar Typhimurium and Yersinia enterocolitica (an enteric bacterium that produces AHLs), SdiA is activated. Although SdiA did not confer an advantage to Salmonella colonization over that of Yersinia, a Salmonella strain engineered to produce AHLs has a colonization advantage over an sdiA mutant in competition assays using mice. The difference in these outcomes could be the result of a transient interaction between Salmonella and Yersinia during coinfection, not allowing a full effect of SdiA signaling to be detected in Salmonella colonization. Meanwhile, constant AHL production in the engineered Salmonella strain allows a colonization phenotype during murine infection. This study indicates for the first time that SdiA is functional during murine infection in the presence of AHLs. Quorum sensing was first described for the regulation of bioluminescence in Vibrio fischeri (9). The luciferase operon in Vibrio fischeri is regulated by two proteins, LuxI, which is responsible for the production of the acyl-homoserine-lactone (AHL) autoinducer, and LuxR, which is activated by this autoinducer to increase transcription of the luciferase operon (6, 7). Since this initial description, homologues of LuxR-LuxI have been identified in other bacteria, and in all of these LuxR-LuxI systems, the bacteria produce an AHL autoinducer, which binds to the LuxR protein and regulates the transcription of several genes involved in a variety of phenotypes. These include the production of antibiotics in Erwinia, motility in Yersinia pseudotuberculosis, and pathogenesis and biofilm formation in Pseudomonas aeruginosa, among others (3, 4, 11). Escherichia coli and Salmonella have a LuxR homologue, SdiA (14), but do not have a luxI gene and do not produce AHLs (8, 13). The E. coli sdiA gene initially was isolated as a regulator of the cell division genes ftsQAZ (14). However, whether SdiA was indeed an AHL sensor was a matter of debate until Michael et al. (8) reported that SdiA is not sensing an autoinducer produced by Salmonella itself but AHLs produced by other bacterial species. This observation makes sense when one considers that unlike Vibrio fischeri, which is in pure culture within the light organ of fish and squids, Salmonella and E. coli are enteric bacteria that colonize the intestine of animals together with many other bacterial species that compose the microbial flora. In this groundbreaking article, Dyszel et al. (5) show that Salmonella SdiA is functional during murine infection when AHLs are present and that SdiA-regulated genes are involved in enhancing Salmonella's colonization of the intestine. Although SdiA is not functional during murine infection with just Salmonella, it is functional during coinfection with an AHL-producing pathogen, Yersinia enterocolitica. This study highlights that coinfection with different pathogens may lead to different outcomes because of cross-chemical communication between different bacterial species.

Production of cell-cell signalling molecules by bacteria isolated from human chronic wounds

To (i) identify chronic wound bacteria and to test their ability to produce acyl-homoserine-lactones (AHLs) and autoinducer-2 (AI-2) cell-cell signalling molecules and (ii) determine whether chronic wound debridement samples might contain these molecules. Partial 16S rRNA gene sequencing revealed the identity of 46 chronic wound strains belonging to nine genera. Using bio-reporter assays, 69.6% of the chronic wound strains were inferred to produce AI-2, while 19.6% were inferred to produce AHL molecules. At least one strain from every genus, except those belonging to the genera Acinetobacter and Pseudomonas, were indicated to produce AI-2. Production of AI-2 in batch cultures was growth-phase dependent. Cross-feeding assays demonstrated that AHLs were produced by Acinetobacter spp., Pseudomonas aeruginosa and Serratia marcescens. Independent from studies of the bacterial species isolated from wounds, AHL and/or AI-2 signalling molecules were detected in 21 of 30 debridement samples of unknown microbial composition. Chronic wound bacteria produce cell-cell signalling molecules. Based on our findings, we hypothesize that resident species generally produce AI-2 molecules, and aggressive transient species associated with chronic wounds typically produce AHLs. Both these classes of cell-cell signals are indicated to be present in human chronic wounds. Interbacterial cell-cell signalling may be an important factor influencing wound development and if this is the case, the presence of AHLs and AI-2 could be used as a predictor of wound severity. Manipulation of cell-cell signalling may provide a novel strategy for improving wound healing.

Biofilm formation and acyl homoserine lactone production in Hafnia alvei isolated from raw milk

The objective of this study was to detect the presence of acyl homoserine lactones (AHLs), signal molecules of the quorum sensing system in biofilm formed by Hafnia alvei strains. It also evaluated the effect of synthetic quorum sensing inhibitors in biofilm formation. AHLs were assayed using well diffusion techniques, thin layer chromatography (TLC) and detection directly in biofilm with biomonitors. The extracts obtained from planktonic and sessile cell of H. alvei induced at least two of three monitor strains evaluated. The presence of AHLs with up to six carbon atoms was confirmed by TLC. Biofilm formation by H. alvei was inhibited by furanone, as demonstrated by 96-well assay of crystal violet in microtitre plates and by scanning electron microscopy. The H. alvei 071 hall mutant was deficient in biofilm formation. All these results showed that the quorum sensing system is probably involved in the regulation of biofilm formation by H. alvei.

Acyl‐homoserine lactones modulate the settlement rate of zoospores of the marine alga Ulva intestinalis via a novel chemokinetic mechanism

Bacteria utilize quorum sensing to regulate the expression of cell density-dependant phenotypes such as biofilm formation and virulence. Zoospores of the marine alga Ulva intestinalis exploit the acyl-homoserine lactone (AHL) quorum sensing system to identify bacterial biofilms for preferential settlement. Here, we demonstrate that AHLs act as strong chemoattractants for Ulva zoospores. Chemoattraction does not involve a chemotactic orientation towards the AHL source. Instead, it occurs through a chemokinesis in which zoospore swimming speed is rapidly decreased in the presence of AHLs. The chemoresponse to AHLs was dependant on the nature of the acyl side chain, with N-(3-oxododecanoyl)-homoserine lactone (30-C12-HSL) being the most effective signal molecule. Mean zoospore swimming speed decreased more rapidly over wild-type biofilms of the marine bacteria Vibrio anguillarum relative to biofilms of the vanM mutant, in which AHL synthesis is disrupted. These data implicate a role for AHL-mediated chemokinesis in the location and preferential settlement of Ulva zoospores on marine bacterial assemblages. Exposure to AHLs did not inhibit the negative phototaxis of Ulva zoospores, indicating that chemoattraction to bacterial biofilms does not preclude the response to a light stimulus in substrate location.