Biomedical subjects

Stephen P Diggle

Publications and source records attributed to Stephen P Diggle.

11 recordsLinked to original sources

PubMed2006

4-quinolone signalling in Pseudomonas aeruginosa: old molecules, new perspectives.

In Pseudomonas aeruginosa, diverse virulence determinants and secondary metabolites are regulated via the action of a hierarchical quorum-sensing system which integrates two chemically distinct classes of signal molecules, the N-acylhomoserine lactones (AHLs) and the 4-quinolones (4Qs). Synthesis of the pseudomonas quinolone signal, 2-heptyl-3-hydroxy-4-quinolone (PQS) depends on the pqsABCDE locus which is responsible for generating multiple 4Qs including 2-heptyl-4-quinolone (HHQ), the immediate PQS precursor. Exported HHQ is taken up by adjacent bacterial cells and converted into PQS by PqsH, a putative mono-oxygenase. In addition, PQS regulates its own production by driving the expression of pqsABCDE through a direct interaction with PqsR (MvfR). PQS regulates diverse target genes including those coding for elastase, rhamnolipid, the PA-IL lectin and pyocyanin via the action of PqsE as well as influencing biofilm development and impacting on cellular fitness. Furthermore, 4Q signalling is not restricted to P. aeruginosa raising the possibility of cross-talk with other related bacterial species which occupy similar ecological niches.

4-Quinolones↗

PubMed2006

Functional genetic analysis reveals a 2-Alkyl-4-quinolone signaling system in the human pathogen Burkholderia pseudomallei and related bacteria.

Pseudomonas aeruginosa synthesizes diverse 2-alkyl-4(1H)-quinolones (AHQs), including the signaling molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), via the pqsABCDE locus. By examining the genome databases, homologs of the pqs genes were identified in other bacteria. However, apart from P. aeruginosa, only Burkholderia pseudomallei and B. thailandensis contained a complete pqsA-E operon (termed hhqA-E). By introducing the B. pseudomallei hhqA and hhqE genes into P. aeruginosa pqsA and pqsE mutants, we show that they are functionally conserved and restore virulence factor and PQS production. B. pseudomallei, B. thailandensis, B. cenocepacia, and P. putida each produced 2-heptyl-4(1H)-quinolone (HHQ), but not PQS. Mutation of hhqA in B. pseudomallei resulted in the loss of AHQ production, altered colony morphology, and enhanced elastase production, which was reduced to parental levels by exogenous HHQ. These data reveal a role for AHQs in bacterial cell-to-cell communication beyond that seen in P. aeruginosa.

Burkholderia pseudomallei↗

PubMed2006

Social evolution theory for microorganisms.

Microorganisms communicate and cooperate to perform a wide range of multicellular behaviours, such as dispersal, nutrient acquisition, biofilm formation and quorum sensing. Microbiologists are rapidly gaining a greater understanding of the molecular mechanisms involved in these behaviours, and the underlying genetic regulation. Such behaviours are also interesting from the perspective of social evolution - why do microorganisms engage in these behaviours given that cooperative individuals can be exploited by selfish cheaters, who gain the benefit of cooperation without paying their share of the cost? There is great potential for interdisciplinary research in this fledgling field of sociomicrobiology, but a limiting factor is the lack of effective communication of social evolution theory to microbiologists. Here, we provide a conceptual overview of the different mechanisms through which cooperative behaviours can be stabilized, emphasizing the aspects most relevant to microorganisms, the novel problems that microorganisms pose and the new insights that can be gained from applying evolutionary theory to microorganisms.

Bacterial Physiological Phenomena↗

PubMed2006

Quorum sensing regulates dpsA and the oxidative stress response in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal human tropical disease. The non-specific DNA-binding protein DpsA plays a key role in protecting B. pseudomallei from oxidative stress mediated, for example, by organic hydroperoxides. The regulation of dpsA expression is poorly understood but one possibility is that it is regulated in a cell population density-dependent manner via N-acylhomoserine lactone (AHL)-dependent quorum sensing (QS) since a lux-box motif has been located within the dpsA promoter region. Using liquid chromatography and tandem mass spectrometry, it was first established that B. pseudomallei strain PP844 synthesizes AHLs. These were identified as N-octanoylhomoserine lactone (C8-HSL), N-(3-oxooctanoyl)homoserine lactone (3-oxo-C8-HSL), N-(3-hydroxyoctanoyl)-homoserine lactone (3-hydroxy-C8-HSL), N-decanoylhomoserine lactone (C10-HSL), N-(3-hydroxydecanoyl) homoserine lactone (3-hydroxy-C10-HSL) and N-(3-hydroxydodecanoyl)homoserine lactone (3-hydroxy-C12-HSL). Mutation of the genes encoding the LuxI homologue BpsI or the LuxR homologue BpsR resulted in the loss of C8-HSL and 3-oxo-C8-HSL synthesis, demonstrating that BpsI was responsible for directing the synthesis of these AHLs only and that bpsI expression and hence C8-HSL and 3-oxo-C8-HSL production depends on BpsR. In bpsI, bpsR and bpsIR mutants, dpsA expression was substantially down-regulated. Furthermore, dpsA expression in Escherichia coli required both BpsR and C8-HSL. bpsIR-deficient mutants exhibited hypersensitivity to the organic hydroperoxide tert-butyl hydroperoxide by displaying a reduction in cell viability which was restored by provision of exogenous C8-HSL (bpsI mutant only), by complementation with the bpsIR genes or by overexpression of dpsA. These data indicate that in B. pseudomallei, QS regulates the response to oxidative stress at least in part via the BpsR/C8-HSL-dependent regulation of DpsA.

4-Butyrolactone↗

PubMed2006

The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa.

LecA (PA-IL) is a cytotoxic lectin and adhesin produced by Pseudomonas aeruginosa which binds hydrophobic galactosides with high specificity and affinity. By using a lecA-egfp translation fusion and immunoblot analysis of the biofilm extracellular matrix, we show that lecA is expressed in biofilm-grown cells. In static biofilm assays on both polystyrene and stainless steel, biofilm depth and surface coverage was reduced by mutation of lecA and enhanced in the LecA-overproducing strain PAO-P47. Biofilm surface coverage by the parent strain, PAO-P47 but not the lecA mutant on steel coupons was also inhibited by growth in the presence of either isopropyl-beta-D-thiogalactoside (IPTG) or p-nitrophenyl-alpha-D-galactoside (NPG). Furthermore, mature wild-type biofilms formed in the absence of these hydrophobic galactosides could be dispersed by the addition of IPTG. In contrast, addition of p-nitrophenyl-alpha-L-fucose (NPF) which has a high affinity for the P. aeruginosa LecB (PA-IIL) lectin had no effect on biofilm formation or dispersal. Planktonic growth of P. aeruginosa PAO1 was unaffected by the presence of IPTG, NPG or NPF, nor was the strain able to utilize these sugars as carbon sources, suggesting that the observed effects on biofilm formation were due to the competitive inhibition of LecA-ligand binding. Similar results were also obtained for biofilms grown under dynamic flow conditions on steel coupons, suggesting that LecA contributes to P. aeruginosa biofilm architecture under different environmental conditions.

Adhesins, Bacterial↗

PubMed2006

Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1.

The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3' position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.

4-Butyrolactone↗

PubMed2005

The MexGHI-OpmD multidrug efflux pump controls growth, antibiotic susceptibility and virulence in Pseudomonas aeruginosa via 4-quinolone-dependent cell-to-cell communication.

In Pseudomonas aeruginosa the production of multiple virulence factors depends on cell-to-cell communication through the integration of N-acylhomoserine lactone (AHL)- and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS)- dependent signalling. Mutation of genes encoding the efflux protein MexI and the porin OpmD from the MexGHI-OpmD pump resulted in the inability to produce N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-c12-hsl) and pqs and a marked reduction in n-butanoyl-L-homoserine lactone levels. Both pump mutants were impaired in growth and exhibited enhanced rather than reduced antibiotic resistance. Provision of exogenous PQS improved growth and restored AHL and virulence factor production as well as antibiotic susceptibility, indicating that the pump mutants retained their capacity to respond to PQS. RT-PCR analysis indicated that expression of the PQS biosynthetic genes, phnA and pqsA, was inhibited when the mutants reached stationary phase, suggesting that the pleiotropic phenotype observed may be due to intracellular accumulation of a toxic PQS precursor. To explore this hypothesis, double mexI phnA (unable to produce anthranilate, the precursor of PQS) and mexI pqsA mutants were constructed; the improved growth of the former suggested that the toxic compound is likely to be anthranilate or a metabolite of it. Mutations in mexI and opmD also resulted in the attenuation of virulence in rat and plant infection models. In plants, addition of PQS restored the virulence of mexI and opmD mutants. Collectively, these results demonstrate an essential function for the MexGHI-OpmD pump in facilitating cell-to-cell communication, antibiotic susceptibility and promoting virulence and growth in P. aeruginosa.

4-Butyrolactone↗

PubMed2004

The Pseudomonas aeruginosa global regulator MvaT specifically binds to the ptxS upstream region and enhances ptxS expression.

Exotoxin A production in Pseudomonas aeruginosa is regulated positively or negatively by several genes. Two such regulatory genes, ptxR and ptxS, which are divergently transcribed from each other, have been described previously. While computer analysis suggested that the ptxR-ptxS intergenic region contains potential binding sites for several regulatory proteins, the mechanism that regulates the expression of either ptxR or ptxS in P. aeruginosa is not known. The presence of a P. aeruginosa protein complex that specifically binds to a segment within this region was determined. In this study the binding region was localized to a 150 bp fragment of the intergenic region and the proteins that constitute the binding complex were characterized as P. aeruginosa HU and MvaT. Recombinant MvaT was purified as a fusion protein (MAL-MvaT) and shown to specifically bind to the ptxR-ptxS intergenic region. A PAO1 isogenic mutant defective in mvaT, PAODeltamvaT, was constructed and characterized. The lysate of PAODeltamvaT failed to bind to the 150 bp probe. The effect of mvaT on ptxS and ptxR expression was examined using real-time PCR experiments. The expression of ptxS was lower in PAODeltamvaT than in PAO1, but no difference was detected in ptxR expression. These results suggest that MvaT positively regulates ptxS expression by binding specifically to the ptxS upstream region.

Bacterial Proteins↗

PubMed2004

Biofilm formation in Pseudomonas aeruginosa: fimbrial cup gene clusters are controlled by the transcriptional regulator MvaT.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen which poses a major threat to long-term-hospitalized patients and individuals with cystic fibrosis. The capacity of P. aeruginosa to form biofilms is an important requirement for chronic colonization of human tissues and for persistence in implanted medical devices. Various stages of biofilm formation by this organism are mediated by extracellular appendages, such as type IV pili and flagella. Recently, we identified three P. aeruginosa gene clusters that were termed cup (chaperone-usher pathway) based on their sequence relatedness to the chaperone-usher fimbrial assembly pathway in other bacteria. The cupA gene cluster, but not the cupB or cupC cluster, is required for biofilm formation on abiotic surfaces. In this study, we identified a gene (mvaT) encoding a negative regulator of cupA expression. Such regulatory control was confirmed by several approaches, including lacZ transcriptional fusions, Northern blotting, and transcriptional profiling using DNA microarrays. MvaT also represses the expression of the cupB and cupC genes, although the extent of the regulatory effect is not as pronounced as with cupA. Consistent with this finding, mvaT mutants exhibit enhanced biofilm formation. Although the P. aeruginosa genome contains a highly homologous gene, mvaU, the repression of cupA genes is MvaT specific. Thus, MvaT appears to be an important regulatory component within a complex network that controls biofilm formation and maturation in P. aeruginosa.

Bacterial Proteins↗

PubMed2003

The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density-dependency of the quorum sensing hierarchy, regulates rhl-dependent genes at the onset of stationary phase and can be produced in the absence of LasR.

In Pseudomonas aeruginosa, diverse exoproduct virulence determinants are regulated via N-acylhomoserine lactone-dependent quorum sensing. Here we show that 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) is also an integral component of the quorum sensing circuitry and is required for the production of rhl-dependent exoproducts at the onset of stationary phase. Analysis of spent P. aeruginosa culture supernatants revealed that PQS is produced at the end of exponential phase in the parent strain and in the late stationary phase of a lasR mutant. Mutants defective in both PQS production (pqsR-) and response (pqsE-) produced substantially reduced levels of exoproducts but retained wild-type N-butanoyl homoserine lactone (C4-HSL) levels. In the wild type, provision of exogenous PQS at the time of inoculation significantly increased PA-IL lectin, pyocyanin and elastase production during early stationary phase and promoted biofilm formation. Exogenous PQS but not PQS derivatives lacking the 3-hydroxy group overcame the cell density but not growth phase-dependent production of exoproducts. PQS also overcame the transcriptional and post-transcriptional repression of lecA (which codes for the PA-IL lectin) mediated via the negative regulators MvaT and RsmA respectively. Increased expression of lecA in the presence of exogenous PQS can be explained partially by increases in RhlR, RpoS and C4-HSL levels. A refined model for quorum sensing in P. aeruginosa is presented.

4-Butyrolactone↗

PubMed2002

Advancing the quorum in Pseudomonas aeruginosa: MvaT and the regulation of N-acylhomoserine lactone production and virulence gene expression.

Pseudomonas aeruginosa regulates the production of many exoproteins and secondary metabolites via a hierarchical quorum-sensing cascade through LasR and RhlR and their cognate signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL) and N-(butanoyl)-L-homoserine lactone (C4-HSL). In this study, we found that transcription of the quorum sensing-regulated genes lecA (coding for PA-IL lectin), lasB (coding for elastase), and rpoS appeared to be growth phase dependent and their expression could not be advanced to the logarithmic phase in cells growing in batch culture by the addition of exogenous C4-HSL and 3O-C12-HSL. To identify novel regulators responsible for this growth phase dependency, a P. aeruginosa lecA::lux reporter strain was subjected to random transposon mutagenesis. A number of mutants affected in lecA expression were found that exhibited altered production of multiple quorum sensing-dependent phenotypes. While some mutations were mapped to new loci such as clpA and mvaT and a putative efflux system, a number of mutations were also mapped to known regulators such as lasR, rhlR, and rpoS. MvaT was identified as a novel global regulator of virulence gene expression, as a mutation in mvaT resulted in enhanced lecA expression and pyocyanin production. This mutant also showed altered swarming ability and production of the LasB and LasA proteases, 3O-C12-HSL, and C4-HSL. Furthermore, addition of exogenous 3O-C12-HSL and C4-HSL to the mvaT mutant significantly advanced lecA expression, suggesting that MvaT is involved in the growth phase-dependent regulation of the lecA gene.

4-Butyrolactone↗