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PubMed2006

Quorum-sensing regulation governs bacterial adhesion, biofilm development, and host colonization in Pantoea stewartii subspecies stewartii.

The phytopathogenic bacterium Pantoea stewartii subsp. stewartii synthesizes stewartan exo/capsular polysaccharide (EPS) in a cell density-dependent manner governed by the EsaI/EsaR quorum-sensing (QS) system. This study analyzes biofilm development and host colonization of the WT and QS regulatory mutant strains of P. stewartii. First, we show that the cell density-dependent synthesis of stewartan EPS, governed by the EsaI/EsaR QS system, is required for proper bacterial adhesion and development of spatially defined, 3D biofilms. Second, a nonvirulent mutant lacking the esaI gene adheres strongly to surfaces and develops densely packed, less structurally defined biofilms in vitro. This strain appears to be arrested in a low cell density developmental mode. Exposure of this strain to exogenous N-acyl-homoserine lactone counteracts this adhesion phenotype. Third, QS mutants lacking the EsaR repressor attach poorly to surfaces and form amorphous biofilms heavily enmeshed in excess EPS. Fourth, the WT strain disseminates efficiently within the xylem, primarily in a basipetal direction. In contrast, the two QS mutant strains remain largely localized at the site of infection. Fifth, and most significantly, epifluorescence microscopic imaging of infected leaf tissue and excised xylem vessels reveals that the bacteria colonize the xylem with unexpected specificity, particularly toward the annular rings and spiral secondary wall thickenings of protoxylem, as opposed to indiscriminate growth to fill the xylem lumen. These observations are significant to bacterial plant pathogenesis in general and may reveal targets for disease control.

Bacterial Adhesion↗

PubMed2006

Identification of the central quorum sensing regulator of virulence in the enteric phytopathogen, Erwinia carotovora: the VirR repressor.

In the Gram-negative phytopathogen, Erwinia carotovora ssp. atroseptica (Eca) virulence depends on the production of a N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) quorum sensing (QS) signal. This work identifies the elusive 'missing link' between QS and virulence in Erwinia. We have identified and characterized a novel regulator of virulence, VirR, in Eca and show that a virR mutation completely restores virulence factor production to an Eca mutant unable to synthesize OHHL. This effect of the virR mutation translates to a restoration of virulence to wild-type levels and thus provides evidence that VirR acts to prevent the production of virulence factors at low cell density. We also show that, in Eca, transcription of virulence genes is controlled by OHHL and that this control is effected through the action of VirR. We also demonstrate that the VirR regulatory pathway is present and functional in both blackleg and soft rotting species of Erwinia.

4-Butyrolactone↗

PubMed2003

Quorum sensing in Candida albicans: probing farnesol's mode of action with 40 natural and synthetic farnesol analogs.

The dimorphic fungus Candida albicans produces extracellular farnesol (3,7,11-trimethyl-2,6,10-dodecatriene-1-ol) which acts as a quorum-sensing molecule (QSM) to suppress filamentation. Of four possible geometric isomers of farnesol, only the E,E isomer possesses QSM activity. We tested 40 natural and synthetic analogs of farnesol for their activity in an N-acetylglucosamine-induced differentiation assay for germ tube formation (GTF). Modified structural features include the head group, chain length, presence or absence of the three double bonds, substitution of a backbone carbon by S, O, N, and Se heteroatoms, presence or absence of a 3-methyl branch, and the bulkiness of the hydrophobic tail. Of the 40 compounds, 22 showed QSM activity by their ability to reduce GTF by 50%. However, even the most active of the analogs tested had only 7.3% of the activity of E,E-farnesol. Structure-activity relationships were examined in terms of the likely presence in C. albicans of a farnesol binding receptor protein.

Antifungal Agents↗

PubMed2006

Structure and inhibition of a quorum sensing target from Streptococcus pneumoniae.

Streptococcus pneumoniae 5'-methylthioadenosine/S-adenosylhomocysteine hydrolase (MTAN) catalyzes the hydrolytic deadenylation of its substrates to form adenine and 5-methylthioribose or S-ribosylhomocysteine (SRH). MTAN is not found in mammals but is involved in bacterial quorum sensing. MTAN gene disruption affects the growth and pathogenicity of bacteria, making it a target for antibiotic design. Kinetic isotope effects and computational studies have established a dissociative S(N)1 transition state for Escherichia coli MTAN, and transition state analogues resembling the transition state are powerful inhibitors of the enzyme [Singh, V., Lee, J. L., Núñez, S., Howell, P. L., and Schramm, V. L. (2005) Biochemistry 44, 11647-11659]. The sequence of MTAN from S. pneumoniae is 40% identical to that of E. coli MTAN, but S. pneumoniae MTAN exhibits remarkably distinct kinetic and inhibitory properties. 5'-Methylthio-Immucillin-A (MT-ImmA) is a transition state analogue resembling an early S(N)1 transition state. It is a weak inhibitor of S. pneumoniae MTAN with a K(i) of 1.0 microM. The X-ray structure of S. pneumoniae MTAN with MT-ImmA indicates a dimer with the methylthio group in a flexible hydrophobic pocket. Replacing the methyl group with phenyl (PhT-ImmA), tolyl (p-TolT-ImmA), or ethyl (EtT-ImmA) groups increases the affinity to give K(i) values of 335, 60, and 40 nM, respectively. DADMe-Immucillins are geometric and electrostatic mimics of a fully dissociated transition state and bind more tightly than Immucillins. MT-DADMe-Immucillin-A inhibits with a K(i) value of 24 nM, and replacing the 5'-methyl group with p-Cl-phenyl (p-Cl-PhT-DADMe-ImmA) gave a K(i) value of 0.36 nM. The inhibitory potential of DADMe-Immucillins relative to the Immucillins supports a fully dissociated transition state structure for S. pneumoniae MTAN. Comparison of active site contacts in the X-ray crystal structures of E. coli and S. pneumoniae MTAN with MT-ImmA would predict equal binding, yet most analogues bind 10(3)-10(4)-fold more tightly to the E. coli enzyme. Catalytic site efficiency is primarily responsible for this difference since k(cat)/K(m) for S. pneumoniae MTAN is decreased 845-fold relative to that of E. coli MTAN.

Amino Acid Sequence↗

PubMedDate not supplied

[Quorum sensing of genes expression--perspective drug target against bacterial pathogenicity].

Bacteria are capable to sense an increase of cell density population and to reply quickly and coordinately by the induction of special sets of genes. This type of the regulation was named Quorum Sensing (QS); it is based on the effect of low-molecular-weight signaling molecules of different nature (autoinducers) which accumulate in the culture at high density of bacterial population and interact with receptor regulatory proteins. QS systems are the global regulators of bacterial genes expression and play a key role in the control of many metabolic processes in cell including the regulation of virulence of bacteria. Here we review the molecular mechanisms of QS systems functioning in bacteria belonging to different taxonomic groups and discuss the potential of QS regulation as a new drug target for the treatment of bacterial infections. At present this approach is accounted as a new alternative strategy of antimicrobial therapy directed on the development of drugs inhibiting QS regulation and active just against pathogenicity of bacteria (antipathogenic drugs). Such a strategy allows to avoid a wide dissemination of resistant forms of pathogenic bacteria and the formation of biofilms increasing in many times the resistance of bacteria to drug preparations.

4-Butyrolactone↗

PubMed2004

Cytokine induction by the P. aeruginosa quorum sensing system during thermal injury.

INTRODUCTION: Pseudomonas aeruginosa causes serious infections in severely burned patients due to its ability to produce numerous virulence factors. The production of most of these factors is controlled by the cell-to-cell communication system called quorum sensing (QS). We have recently shown that several proinflammatory and hematopoietic cytokines are produced during infection of the burn wound with P. aeruginosa strain PAO1. Most of these cytokines were not produced during either thermal injury or P. aeruginosa infection alone. MATERIALS AND METHODS AND RESULTS: In this study, we tried to determine if the QS systems play a role in the production of cytokines during P. aeruginosa infection of burn wounds. This was accomplished using the murine model of thermal injury, the P. aeruginosa strain PAO1 and its QS defective mutant (PAO-JP2), and the Multi-probe RNase protection assay. The mRNA for TNF-alpha, IL-6, TGF-beta, and G-CSF was detected within the skin of PAO1 infected/thermally injured mice. In contrast, the expression of these cytokines was not detected in PAO-JP2 infected/thermally injured mice. In comparison with the parent strain, PAO-JP2 was not defective either in its growth or in its spread within the thermally injured skin. A complementation experiment, using a plasmid that carries the intact QS gene, was conducted to confirm these results. In the presence of the complementing plasmid, PAO-JP2 produced the mRNA for the above cytokines. CONCLUSIONS: These results suggest that: 1) the QS system is involved in the induction of cytokine expression during P. aeruginosa infection of burn wounds; and 2) this effect may be caused by either a component of the QS system or a QS-controlled virulence factor.

Animals↗

PubMed2005

Garlic blocks quorum sensing and promotes rapid clearing of pulmonary Pseudomonas aeruginosa infections.

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis patients. P. aeruginosa colonizes the lungs by forming biofilm microcolonies throughout the lung. Quorum sensing (QS) renders the biofilm bacteria highly tolerant to otherwise lethal doses of antibiotics, and protects against the bactericidal activity of polymorphonuclear leukocytes (PMNs). It has been previously demonstrated that QS is inhibited by garlic extract. In this study, the synergistic effects of garlic and tobramycin, and PMNs activities have been evaluated. P. aeruginosa was grown in vitro in continuous-culture once-through flow chambers with and without garlic extract. The garlic-treated biofilms were susceptible to both tobramycin and PMN grazing. Furthermore, the PMNs showed an increase in respiratory burst activation, when incubated with the garlic-treated biofilm. Garlic extract was administered as treatment for a mouse pulmonary infection model. Mice were treated with garlic extract or placebo for 7 days, with the initial 2 days being prophylactic before P. aeruginosa was instilled in the left lung of the mice. Bacteriology, mortality, histopathology and cytokine production were used as indicators. The garlic treatment initially provoked a higher degree of inflammation, and significantly improved clearing of the infecting bacteria. The results indicate that a QS-inhibitory extract of garlic renders P. aeruginosa sensitive to tobramycin, respiratory burst and phagocytosis by PMNs, as well as leading to an improved outcome of pulmonary infections.

Animals↗

PubMed1999

Quorum sensing in Burkholderia cepacia: identification of the LuxRI homologs CepRI.

Burkholderia cepacia has emerged as an important pathogen in patients with cystic fibrosis. Many gram-negative pathogens regulate the production of extracellular virulence factors by a cell density-dependent mechanism termed quorum sensing, which involves production of diffusible N-acylated homoserine lactone signal molecules, called autoinducers. Transposon insertion mutants of B. cepacia K56-2 which hyperproduced siderophores on chrome azurol S agar were identified. One mutant, K56-R2, contained an insertion in a luxR homolog that was designated cepR. The flanking DNA region was used to clone the wild-type copy of cepR. Sequence analysis revealed the presence of cepI, a luxI homolog, located 727 bp upstream and divergently transcribed from cepR. A lux box-like sequence was identified upstream of cepI. CepR was 36% identical to Pseudomonas aeruginosa RhlR and 67% identical to SolR of Ralstonia solanacearum. CepI was 38% identical to RhlI and 64% identical to SolI. K56-R2 demonstrated a 67% increase in the production of the siderophore ornibactin, was protease negative on dialyzed brain heart infusion milk agar, and produced 45% less lipase activity in comparison to the parental strain. Complementation of a cepR mutation restored parental levels of ornibactin and protease but not lipase. An N-acylhomoserine lactone was purified from culture fluids and identified as N-octanoylhomoserine lactone. K56-I2, a cepI mutant, was created and shown not to produce N-octanoylhomoserine lactone. K56-I2 hyperproduced ornibactin and did not produce protease. These data suggest both a positive and negative role for cepIR in the regulation of extracellular virulence factor production by B. cepacia.

Amino Acid Sequence↗

PubMed2003

[Clinical strains of Burkholderia cepacia: characteristic and detection of the components in the quorum sensing regulatory system].

Described in the paper are characteristics of B. cepacia clinical strains isolated from patients at Moscow hospitals. The strains were investigated for the presence of proteolytic, chitinolytic, hemolytic and lipase activities as well as for presence of components of the "Quorum sensing" gene activity regulatory system by using biological test-systems and in the polymerase chain reaction with primers to genes cepI and cepR.

Anti-Bacterial Agents↗

PubMedDate not supplied

[Quorum-sensing regulation in soil pseudomonads].

228 strains of soil and rhizosphere pseudomonads isolated in different geographic zones were screened, with the use of two tester systems, for the capacity to produce N-acetyl-homoserine lactones (AHLs), which are autoinducers involved in quorum-sensing (QS) regulation. AHL production was found in 11.4% of the strains investigated. In five Pseudomonas chlororaphis strains shown to be active AHL producers and chosen for further study, PCR identified two QS systems that involved the phzI, phzR, csaI, and csaR genes; this finding suggests the conservative nature of these regulation systems in P. chlororaphis. Strain P. chlororaphis 449, chosen as a model object and studied in greater detail, produced three AHL species including N-butanoyl-homoserine lactone and N-hexanoyl-homoserine lactone. This strain produced three types of phenazine antibiotics, as well as siderophores and cyanide; it also exhibited antagonistic properties toward a wide spectrum of phytopathogenic fungi. The phzI and csaI genes, coding for synthases of AHLs of two types, were cloned and sequenced; mutants with knocked-out phzI and csal genes were obtained. With the use of transposon mutagenesis and the gene substitution method, mutations were obtained in the global expression regulator genes gacS, coding for the GacA-GacS regulation system kinase, and rpoS, coding for the sigma S subunit of RNA polymerase. The effect of these mutations on the AHL synthesis and on the regulation of various metabolic processes in P. chlororaphis was studied.

4-Butyrolactone↗

PubMed2005

Elevated temperature enhances virulence of Erwinia carotovora subsp. carotovora strain EC153 to plants and stimulates production of the quorum sensing signal, N-acyl homoserine lactone, and extracellular proteins.

Erwinia carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, and E. carotovora subsp. carotovora produce high levels of extracellular enzymes, such as pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt), and the quorum-sensing signal N-acyl-homoserine lactone (AHL) at 28 degrees C. However, the production of these enzymes and AHL by these bacteria is severely inhibited during growth at elevated temperatures (31.2 degrees C for E. carotovora subsp. atroseptica and 34.5 degrees C for E. carotovora subsp. betavasculorum and most E. carotovora subsp. carotovora strains). At elevated temperatures these bacteria produce high levels of RsmA, an RNA binding protein that promotes RNA decay. E. carotovora subsp. carotovora strain EC153 is an exception in that it produces higher levels of Pel, Peh, Cel, and Prt at 34.5 degrees C than at 28 degrees C. EC153 also causes extensive maceration of celery petioles and Chinese cabbage leaves at 34.5 degrees C, which correlates with a higher growth rate and higher levels of rRNA and AHL. The lack of pectinase production by E. carotovora subsp. carotovora strain Ecc71 at 34.5 degrees C limits the growth of this organism in plant tissues and consequently impairs its ability to cause tissue maceration. Comparative studies with ahlI (the gene encoding a putative AHL synthase), pel-1, and peh-1 transcripts documented that at 34.5 degrees C the RNAs are more stable in EC153 than in Ecc71. Our data reveal that overall metabolic activity, AHL levels, and mRNA stability are responsible for the higher levels of extracellular protein production and the enhanced virulence of EC153 at 34.5 degrees C compared to 28 degrees C.

4-Butyrolactone↗

PubMed2002

Quorum sensing and production of autoinducer-2 in Campylobacter spp., Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium in foods.

Autoinducer molecules are utilized by gram-negative and gram-positive bacteria to regulate density-dependent gene expression by a mechanism known as quorum sensing. PCR and DNA sequencing results showed that Campylobacter jejuni and Campylobacter coli possessed luxS, which is responsible for autoinducer-2 (AI-2) production. Using a Vibrio harveyi luminescence assay, the production of AI-2 was observed in milk, chicken broth, and brucella broth by C. coli, C. jejuni, Salmonella enterica serovar Typhimurium, and Escherichia coli O157:H7 under different conditions.

Amino Acid Sequence↗

PubMed2004

Boron binding with the quorum sensing signal AI-2 and analogues.

[reaction: see text] The unstable bacterial metabolic product, DPD, and the related natural product, laurencione, are shown to have a high affinity for borate complexation, through the hydrated analogue. The boron complex of DPD is Vibrio harveyi AI-2, an interspecies quorum sensing signal in bacteria, and an affinity column with a borate resin is effective in providing the first method for concentrating and purifying V. harveyi AI-2 from the biosynthetic product.

Boron↗

PubMed1998

Activity of the quorum-sensing regulator TraR of Agrobacterium tumefaciens is inhibited by a truncated, dominant defective TraR-like protein.

Horizontal transfer of Agrobacterium tumefaciens tumour-inducing plasmids requires opines, which are released from plant tumours as nutrients for the bacteria. The opine octopine causes synthesis of the quorum-sensing TraR protein, which activates several tra promoters in the presence of a pheromone called Agrobacterium autoinducer (AAI). A gene, traS, was previously found on the same Ti plasmid in an operon that directs the uptake of mannopine, another opine. TraS strongly resembles TraR but lacks a DNA-binding module. TraS did not activate a TraR-dependent promoter and blocked TraR function, probably by forming inactive heteromultimers. Expression of traS was induced by mannopine, although this induction was strongly inhibited by the favoured catabolites succinate, glutamine and tryptone. Mannopine inhibited conjugation in a TraS-dependent fashion, and artificial overexpression of TraS also inhibited conjugation. Favoured catabolites restored tra gene expression in wild-type strains but not in strains that overexpress TraS. Downstream of traS is a gene encoding a truncated, defective chemoreceptor whose expression abolished chemotaxis.

Agrobacterium tumefaciens↗

PubMed2004

Hydrophobic interactions drive ligand-receptor recognition for activation and inhibition of staphylococcal quorum sensing.

Two-component systems represent the most widely used signaling paradigm in living organisms. Encoding the prototypical two-component system in Gram-positive bacteria, the staphylococcal agr (accessory gene regulator) operon uses a polytopic receptor, AgrC, activated by an autoinducing peptide (AIP), to coordinate quorum sensing with the global synthesis of virulence factors. The agr locus has undergone evolutionary divergence, resulting in the formation of several distinct inter- and intraspecies specificity groups, such that most cross-group AIP-receptor interactions are mutually inhibitory. We have exploited this natural diversity by constructing and analyzing AgrC chimeras generated by exchange of intradomain segments between receptors of different agr groups. Functional chimeras fell into three general classes: receptors with broadened specificity, receptors with tightened specificity, and receptors that lack activation specificity. Testing of these chimeric receptors against a battery of AIP analogs localized the primary ligand recognition site to the receptor distal subdomain and revealed that the AIPs bind primarily to a putative hydrophobic pocket in the receptor. This binding is mediated by a highly conserved hydrophobic patch on the AIPs and is an absolute requirement for interactions in self-activation and cross-inhibition of the receptors. It is suggested that this recognition scheme provides the fundamental basis for agr activation and interference.

Amino Acid Sequence↗

PubMed2006

The quorum-sensing negative regulator RsaL of Pseudomonas aeruginosa binds to the lasI promoter.

A mutation in the rsaL gene of Pseudomonas aeruginosa produces dramatically higher amounts of N-acyl homoserine lactone with respect to the wild type, highlighting the key role of this negative regulator in controlling quorum sensing (QS) in this opportunistic pathogen. The DNA binding site of the RsaL protein on the rsaL-lasI bidirectional promoter partially overlaps the binding site of the LasR protein, consistent with the hypothesis that RsaL and LasR could be in binding competition on this promoter. This is the first direct demonstration that RsaL acts as a QS negative regulator by binding to the lasI promoter.

Bacterial Proteins↗

PubMed2003

Dimerization of the quorum sensing regulator RhlR: development of a method using EGFP fluorescence anisotropy.

Of considerable interest in the biology of pathogenic bacteria are the mechanisms of intercellular signalling that can lead to the formation of persistent infections. In this article, we have examined the intracellular behaviour of a Pseudomonas aeruginosa quorum sensing regulator RhlR believed to be important in this process. We have further examined the modulation of this behaviour in response to various auto-inducers. For these measurements, we have developed an assay based on the fluorescence anisotropy of EGFP fusion proteins that we use to measure protein-protein interactions in vivo. We show that the transcriptional regulator, RhlR, expressed as an EGFP fusion protein in Escherichia coli, forms a homodimer. This homodimer can be dissociated into monomers by the auto-inducer N-(3-oxododecanoyl)-l-homoserine lactone (3O-C12-HSL) whereas N-(butanoyl)-l-homoserine lactone (C4-HSL) has little effect. These observations are of particular interest as RhlR modulation of gene expression depends on the presence of C4-HSL, whereas 3O-C12-HSL modulates the expression of genes regulated by LasR. These observations thus provide a framework for understanding the regulatory network that links the various different QS regulators in P. aeruginosa. Furthermore, the technique we have developed should permit the study of numerous protein/protein or protein/nucleic acid interactions in vivo and so shed light on natural protein function.

Bacterial Proteins↗

PubMed2005

Quorum sensing in Clostridium difficile: analysis of a luxS-type signalling system.

The increasing incidence of Clostridium difficile-associated disease, and the problems associated with its control, highlight the need for additional countermeasures. The attenuation of virulence through the blockade of bacterial cell-to-cell communication (quorum sensing) is one potential therapeutic target. Preliminary studies have shown that C. difficile produces at least one potential signalling molecule. Through the molecule's ability to induce bioluminescence in a Vibrio harveyi luxS reporter strain, it has been shown to correspond to autoinducer 2 (AI-2). In keeping with this observation, a homologue of luxS has been identified in the genome of C. difficile. Adjacent to luxS(Cd) a potential transcriptional regulator and sensor kinase, rolA and rolB, have been located. RT-PCR has been used to confirm the genetic organization of the luxS(Cd) locus. While AI-2 production has not been blocked so far using antisense technology, AI-2 levels could be modulated by controlling expression of the putative transcriptional regulator rolA. RolA, therefore, acts as a negative regulator of AI-2 production. Finally, it has been shown that the exogenous addition of AI-2 or 4-hydroxy-5-methyl-3(2H) furanone has no discernible effect on the production of toxins by C. difficile.

Amino Acid Sequence↗