Quorum Sensing in <Emphasis Type="Italic">Aeromonas</Emphasis> Species Isolated from Patients in Malaysia

Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C₆. Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.     

Occurrence of N-acyl-L-homoserine lactones in extracts of some Gram-negative bacteria evaluated by gas chromatography-mass spectrometry

Acylated homoserine lactones (AHLs) are self-generated signal molecules that mediate population density-dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria. These signal molecules diffuse from bacterial cells and accumulate in the medium as a function of cell growth. In selected foods AHLs contribute to product spoilage. As different bacterial species produce AHL analogs that differ in length of the N-acyl chain, ranging from 4 to 14 carbons and in the substitution at the C-3 position of the side chain (i.e., oxo or hydroxyl group), the suitability and applicability of a gas chromatography-mass spectrometry direct method for characterizing trace amounts of AHLs was evaluated using N-heptanoyl-homoserine lactone as internal standard. Crude cell-free supernatants of bacterial cultures of Aeromonas hydrophila, Aeromonas salmonicida, Pseudomonas aeruginosa, Pseudomonas fluorescens, Yersinia enterocolitica, and Serratia liquefaciens were screened for AHL production in selected ion monitoring mode, using the prominent fragment at m/z 143. The observed profiles of distinguishable N-acyl-homoserine lactones occurring in bacterial extracts were compared and discussed. The presence of a labile 3-oxo-hexanoylhomoserine lactone was evidenced but serious difficulties arose in estimating its concentration as thermal degradation occurs during the gas chromatographic separation. Its electron impact mass spectra was, however, given and interpreted.     

Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide-producing species of Halomonas

Some members of the moderately halophilic genus Halomonas, such as H. eurihalina, H. maura, H. ventosae and H. anticariensis, produce exopolysaccharides with applications in many industrial fields. We report here that these four species also produce autoinducer molecules that are involved in the cell-to-cell signaling process known as quorum sensing. By using the N-acyl homoserine lactone (AHL) indicator strains Agrobacterium tumefaciens NTL4 (pZRL4) and Chromobacterium violaceum CV026, we discovered that all the Halomonas strains examined synthesize detectable AHL signal molecules. The synthesis of these compounds was growth-phase dependent and maximal activity was reached during the late exponential to stationary phases. One of these AHLs seems to be synthesized only in the stationary phase. Some of the AHLs produced by H. anticariens FP35^T were identified by gas chromatography/mass spectrometry and electrospray ionization tandem mass spectrometry as N-butanoyl homoserine lactone (C₄-HL), N-hexanoyl homoserine lactone (C₆-HL), N-octanoyl homoserine lactone (C₈-HL) and N-dodecanoyl homoserine lactone (C₁₂-HL). This study suggests that quorum sensing may also play an important role in extreme environments.     

A probable aculeacin A acylase from the Ralstonia solanacearum GMI1000 is N-acyl-homoserine lactone acylase with quorum-quenching activity

Background  

The infection and virulence functions of diverse plant and animal pathogens that possess quorum sensing systems are regulated by N-acylhomoserine lactones (AHLs) acting as signal molecules. AHL-acylase is a quorum quenching enzyme and degrades AHLs by removing the fatty acid side chain from the homoserine lactone ring of AHLs. This blocks AHL accumulation and pathogenic phenotypes in quorum sensing bacteria.     

Investigation of N-acyl homoserine lactone (AHL) molecule production in Gram-negative bacteria isolated from cooling tower water and biofilm samples

In this study, 99 Gram-negative rod bacteria were isolated from cooling tower water, and biofilm samples were examined for cell-to-cell signaling systems, N-acyl homoserine lactone (AHL) signal molecule types, and biofilm formation capacity. Four of 39 (10 %) strains isolated from water samples and 14 of 60 (23 %) strains isolated from biofilm samples were found to be producing a variety of AHL signal molecules. It was determined that the AHL signal molecule production ability and the biofilm formation capacity of sessile bacteria is higher than planktonic bacteria, and there was a statistically significant difference between the AHL signal molecule production of these two groups (p?<?0.05). In addition, it was found that bacteria belonging to the same species isolated from cooling tower water and biofilm samples produced different types of AHL signal molecules and that there were different types of AHL signal molecules in an AHL extract of bacteria. In the present study, it was observed that different isolates of the same strains did not produce the same AHLs or did not produce AHL molecules, and bacteria known as AHL producers did not produce AHL. These findings suggest that detection of signal molecules in bacteria isolated from cooling towers may contribute to prevention of biofilm formation, elimination of communication among bacteria in water systems, and blockage of quorum-sensing controlled virulence of these bacteria.     

Inhibition of Quorum Sensing Mediated Virulence Factors Production in Urinary Pathogen Serratia marcescens PS1 by Marine Sponges

The focal intent of this study was to find out an alternative strategy for the antibiotic usage against bacterial infections. The quorum sensing inhibitory (QSI) activity of marine sponges collected from Palk Bay, India was evaluated against acyl homoserine lactone (AHL) mediated violacein production in Chromobacterium violaceum (ATCC 12472), CV026 and virulence gene expressions in clinical isolate Serratia marcescens PS1. Out of 29 marine sponges tested, the methanol extracts of Aphrocallistes bocagei (TS 8), Haliclona (Gellius) megastoma (TS 25) and Clathria atrasanguinea (TS 27) inhibited the AHL mediated violacein production in C. violaceum (ATCC 12472) and CV026. Further, these sponge extracts inhibited the AHL dependent prodigiosin pigment, virulence enzymes such as protease, hemolysin production and biofilm formation in S. marcescens PS1. However, these sponge extracts were not inhibitory to bacterial growth, which reveals the fact that the QSI activity of these extracts was not related to static or killing effects on bacteria. Based on the obtained results, it is envisaged that the marine sponges could pave the way to prevent quorum sensing (QS) mediated bacterial infections.     

Identification of virulence markers in clinically relevant strains of Acinetobacter genospecies

Nine Acinetobacter strains from patients and hospital environment were analyzed for virulence markers, quorum sensing signal production, and the presence of luxI and luxR genes. The strains had several properties in common: growth in iron limited condition, biofilm formation, and no active protease secretion. Significantly higher catechol production was determined in patient isolates (P < 0.03), but other invasiveness markers, such as lipase secretion, amount of biofilm, cell motility, antibiotic resistance, and hemolysin production, showed large variability. Notably, all members of the so-called A. calcoaceticus-A. baumannii complex, regardless of whether the source was a patient or environmental, secreted mediumto long-chain N-acyl homoserine lactones (AHL) and showed blue light inhibition of cell motility. In these strains, a luxI homologue with a homoserine lactone synthase domain and a luxR putative regulator displaying the typical AHL binding domain were identified.     

N-butanoyl-l-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit

Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low.     

Quorum sensing, virulence and secondary metabolite production in plant soft-rotting bacteria

Quorum sensing describes the ability of bacteria to sense their population density and respond by modulating gene expression. In the plant soft-rotting bacteria, such as Erwinia, an arsenal of plant cell wall-degrading enzymes is produced in a cell density-dependent manner, which causes maceration of plant tissue. However, quorum sensing is central not only to controlling the production of such destructive enzymes, but also to the control of a number of other virulence determinants and secondary metabolites. Erwinia synthesizes both N-acylhomoserine lactone (AHL) and autoinducer-2 types of quorum sensing signal, which both play a role in regulating gene expression in the phytopathogen. We review the models for AHL-based regulation of carbapenem antibiotic production in Erwinia. We also discuss the importance of quorum sensing in the production and secretion of virulence determinants by Erwinia, and its interplay with other regulatory systems.     

Heterologous overexpression of quorum-sensing regulators to study cell-density-dependent phenotypes in a symbiotic plant bacterium <Emphasis Type="Italic">Mesorhizobium huakuii</Emphasis>

Quorum-sensing is widespread among many prokaryotic lineages. In order to investigate quorum regulation in the plant bacterium Mesorhizobium huakuii which produces an N-acyl homoserine lactone (AHL) quorum signal, the Agrobacterium quorum-sensing regulator TraR was heterologously expressed in this bacterium. The resulting strains showed reduced AHL production in the supernatant compared to wild-type, but similar intracellular levels of AHLs were detected, suggesting that M. huakuii AHLs can be bound to intracellular TraR proteins and thus become unavailable for its own quorum systems. M. huakuii overexpressing TraR formed thinner biofilms than the wild-type, suggesting a role played by quorum-sensing in biofilm formation.Hui Wang and Zengtao Zhong contributed equally to this work.     

PpoR is a conserved unpaired LuxR solo of Pseudomonas putida which binds N-acyl homoserine lactones

Background  

Only a small number of Pseudomonas putida strains possess the typical N-acyl homoserine lactone quorum sensing system (AHL QS) that consists of a modular LuxR family protein and its cognate LuxI homolog that produces the AHL signal. Moreover, AHL QS systems in P. putida strains are diverse in the type of AHLs they produce and the phenotypes that they regulate.     

Molecular identification of aiiA homologous gene from endophytic Enterobacter species and in silico analysis of putative tertiary structure of AHL-lactonase

The aiiA homologous gene known to encode AHL- lactonase enzyme which hydrolyze the N-acylhomoserine lactone (AHL) quorum sensing signaling molecules produced by Gram negative bacteria. In this study, the degradation of AHL molecules was determined by cell-free lysate of endophytic Enterobacter species. The percentage of quorum quenching was confirmed and quantified by HPLC method (p < 0.0001). Amplification and sequence BLAST analysis showed the presence of aiiA homologous gene in endophytic Enterobacter asburiae VT65, Enterobacter aerogenes VT66 and Enterobacter ludwigii VT70 strains. Sequence alignment analysis revealed the presence of two zinc binding sites, "HXHXDH" motif as well as tyrosine residue at the position 194. Based on known template available at Swiss-Model, putative tertiary structure of AHL-lactonase was constructed. The result showed that novel endophytic strains of Enterobacter genera encode the novel aiiA homologous gene and its structural importance for future study.     

Inhibition of Quorum Sensing in Serratia marcescens AS-1 by Synthetic Analogs of N-Acylhomoserine Lactone

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C₆-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C₉-CPA), had a strong inhibitory effect on prodigiosin production. C₉-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C₉-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C₆-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C₉-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

Acyl-homoserine lactone-dependent eavesdropping promotes competition in a laboratory co-culture model

Many Proteobacteria use acyl-homoserine lactone (AHL)-mediated quorum sensing to activate the production of antibiotics at high cell density. Extracellular factors like antibiotics can be considered public goods shared by individuals within a group. Quorum-sensing control of antibiotic production may be important for protecting a niche or competing for limited resources in mixed bacterial communities. To begin to investigate the role of quorum sensing in interspecies competition, we developed a dual-species co-culture model using the soil saprophytes Burkholderia thailandensis (Bt) and Chromobacterium violaceum (Cv). These bacteria require quorum sensing to activate the production of antimicrobial factors that inhibit growth of the other species. We demonstrate that quorum-sensing-dependent antimicrobials can provide a competitive advantage to either Bt or Cv by inhibiting growth of the other species in co-culture. Although the quorum-sensing signals differ for each species, we show that the promiscuous signal receptor encoded by Cv can sense signals produced by Bt, and that this ability to eavesdrop on Bt can provide Cv an advantage in certain situations. We use an in silico approach to investigate the effect of eavesdropping in competition, and show conditions where early activation of antibiotic production resulting from eavesdropping can promote competitiveness. Our work supports the idea that quorum sensing is important for interspecies competition and that promiscuous signal receptors allow eavesdropping on competitors in mixed microbial habitats.     

Pseudomonas corrugata contains a conserved N-acyl homoserine lactone quorum sensing system; its role in tomato pathogenicity and tobacco hypersensitivity response

Pseudomonas corrugata is a phytopathogenic bacterium, causal agent of tomato pith necrosis, yet it is an ubiquitous bacterium that is part of the microbial community in the soil and in the rhizosphere of different plant species. Although it is a very heterogeneous species, all the strains tested were able to produce short chain acyl homoserine lactone (AHL) quorum sensing signal molecules. The main AHL produced was N-hexanoyl-L-homoserine lactone (C(6)-AHL). An AHL quorum sensing system, designated PcoI/PcoR, was identified and characterized. The role of the quorum sensing system in the expression of a variety of traits was evaluated. Inactivation of pcoI abolished the production of AHLs. The pcoR mutant, but not the pcoI mutant, was impaired in swarming, unable to cause a hypersensitivity response on tobacco and resulted in a reduced tomato pith necrosis phenotype. The pcoI mutant showed a reduced antimicrobial activity against various fungi and bacteria when assayed on King's B medium. These results demonstrate that the AHL quorum sensing in Ps. corrugata regulates traits that contribute to virulence, antimicrobial activity and fitness. This is the first report of genes of Ps. corrugata involved in the disease development and biological control activity.     

Detection of Quorum Sensing Signal Molecules in Edwardsiella ictaluri Ei-151

Edwardsiella ictaluri is a Gram-negative pathogenic bacterium in the family Enterobacteriaceae that causes enteric septicemia of catfish, which has become a significant problem in the aquaculture of striped catfish (Pangasianodon hypophthalmus) in Vietnam. In this study, a bacterium designated as Ei-151 was isolated from diseased striped catfish and proved to be virulent. Based on 16S rDNA sequencing and phenotypic tests, the pathogenic bacterium was identified as Edw. ictaluri. The presence of quorum sensing signal molecules in Edw. ictaluri Ei-151 was detected with different biosensor strains. The results showed that Ei-151 produced at least three kinds of acylated homoserine lactone (AHL) signal molecules as detected with the biosensor Agrobacterium tumefaciens KYC55, and the AHLs fingerprint was similar to that of Edw. tarda. During its entire growth, the levels of AHLs and autoinducer-2 produced by Ei-151 peaked at the stationary phase (OD₆₀₀ 1.8), which suggested that both of them may function at the stationary phase. No Cholerae autoinducer-1-like activity (including Edw. ictaluri LMG7860^T) was detected.     

N-Acylated homoserine lactone production and involvement in the biodegradation of aromatics by an environmental isolate of Pseudomonas aeruginosa

N-Acyl homoserine lactone (AHL) is a widespread quorum sensing signal molecule in Gram-negative bacteria and has an important role in many biological processes. However, it is still poorly understood whether or not AHL is present in pollutant treatment processes and further, what its role is in biodegradation processes. In this work, an environmental isolate of Pseudomonas aeruginosa CGMCC 1.860 that is an aromatic degrader and AHL producer was selected. The AHL plate bioassay indicated that AHL was produced by this strain during biodegradation of aromatic compounds including phenol, benzoate, p-hydroxy-benzoate, salicylate, and naphthalene. The AHLs were identified as N-butyryl-l-homoserine lactone (BHL) and N-hexanoyl-l-homoserine lactone (HHL) by using thin layer chromatography (TLC) and high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (HPLC–APCI-MS/MS) analyses. Furthermore, phenol biodegradation was improved by exogenously added AHL extracts or by endogenously over-produced AHLs, repressed by abolishment of AHLs production, and not affected by the addition of extracts without AHLs. The results indicated that AHL was involved in the process of biodegradation of pollutants.     

Identification of Quorum-Quenching N-Acyl Homoserine Lactonases from Bacillus Species

A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.     

Quorum quenching enzyme activity is widely conserved in the sera of mammalian species

Acyl-homoserine lactone (AHL) quorum sensing signals play a key role in synchronizing virulence gene expression in Pseudomonas aeruginosa, which could cause fatal bloodstream infections. We showed that AHL inactivation activity, albeit with variable efficiency, was conserved in the serum samples of all the 6 tested mammalian animals. High-performance liquid chromatography and mass spectrometry analyses revealed that mammalian sera had a lactonase-like enzyme(s), which hydrolyzed the lactone ring of AHL to produce acyl homoserine, with enzyme properties reminiscent of paraoxonases (PONs). We further showed that the animal cell lines expressing three mouse PON genes, respectively, displayed strong AHL degradation activities.