Biomedical subjects

Paul Williams

Publications and source records attributed to Paul Williams.

At least 19 recordsLinked to original sources

PubMed2006

Comprehensive profiling of N-acylhomoserine lactones produced by Yersinia pseudotuberculosis using liquid chromatography coupled to hybrid quadrupole-linear ion trap mass spectrometry.

A method for the comprehensive profiling of the N-acylhomoserine lactone (AHL) family of bacterial quorum-sensing molecules is presented using liquid chromatography (LC) coupled to hybrid quadrupole-linear ion trap (QqQLIT) mass spectrometry. Information-dependent acquisition (IDA), using triggered combinations of triple-quadrupole and linear ion trap modes in the same LC-MS/MS run, was used to simultaneously screen, quantify and identify multiple AHLs in a single sample. This MS method uses common AHL fragment ions attributed to the homoserine moiety and the 3-oxo-, 3-hydroxy- or unsubstituted acyl side chains, to identify unknown AHLs in cell-free culture supernatants in an unbiased manner. This LC-MS technique was applied to determine the relative molar ratios of AHLs produced by Yersinia pseudotuberculosis and the consequences of inactivating by mutation either or both of the AHL synthase genes (ypsI and ytbI) on AHL profile and concentration. The Y. pseudotuberculosis wild type but not the ypsI ytbI double mutant produced at least 24 different AHLs with acyl chains ranging from C4 to C15 with or without 3-oxo or 3-hydroxy substituents. YtbI, in contrast to YpsI, could direct the synthesis of all of the AHLs identified. The most abundant and hence most biologically relevant Y. pseudotuberculosis AHLs were found to be the 3-oxo-substituted C6, C7 and C8 AHLs and the unsubstituted C6 and C8 compounds. The LC-QqQLIT methodology is broadly applicable to quorum-sensing signal molecule analysis and can provide comprehensive AHL profiles and concentrations from a single sample and simultaneously collect confirmatory spectra for each AHL identified.

4-Butyrolactone↗

PubMed2006

Foot-and-mouth disease - quantification and size distribution of airborne particles emitted by healthy and infected pigs.

There is strong evidence to suggest that foot-and-mouth disease (FMD) can be transmitted by airborne virus up to many kilometres from a virus source. Atmospheric dispersion models are often used to predict where this disease might spread. This study investigated whether FMD virus (FMDV) aerosol has specific characteristics which need to be taken into consideration in these models. The characteristics and infectiousness of particles emitted by 12 pigs have been studied pre- and post-infection with O UKG 2001 FMDV. Aerosol generated by individual pigs was found log normally distributed in the range 0.015-20.0microm with concentrations between 1000 and 10000cm(-3) at the smallest size and <1cm(-3) above 10microm. No differences in either the total number of particles produced or their size distribution were detected between uninfected and infected pigs. However, a correlation between aerosol concentration and animal activity was found with a more active pig producing significantly greater concentrations than those that were less active. Viable virus was found up to a maximum of 6.3 log TCID(50)/24h/animal. The virus was distributed almost equally across the three size ranges; <3, 3-6 and >6microm. No correlation could be established between the production of virus and animal activity. In general the production of airborne virus closely followed the detection of viraemia in the blood and the presence of clinical symptoms. However, in one instance a pig excreted as much airborne virus as the other animals in the study, but with less virus detected in its blood. The results suggest that there is little merit in including a sophisticated virus release pattern based on physical activity periods or FMDV aerosol size spectrum, together with the appropriate dry deposition calculations, in models used to predict airborne spread of FMD. An estimate of the total daily virus production based on the clinical assessment of disease and virus strain is sufficient as input.

Air Microbiology↗

PubMed2006

Regulatory roles of spnT, a novel gene located within transposon TnTIR.

The transposon TnTIR contains spnIR quorum-sensing system regulating sliding motility and the production of nuclease, biosurfactant, and prodigiosin in Serratia marcescens. Within TnTIR, a gene named spnT is upstream of and co-transcribed with spnI. SpnT is a cytoplasmic protein and its level peaks during early stationary phase. spnT over-expression resulted in inhibition of sliding motility and synthesis of prodigiosin, and biosurfactant similar to spnR. spnT but not spnR over-expression induced cell elongation and aberrant DNA replication in S. marcescens and Escherichia coli strains. In comparison with wild-type E. coli strain, over-expression of spnT in an E. coli priA and dnaC double-mutant strain did not lead to the aberrant cell morphology phenotypes, suggesting SpnT may act through the recombination-dependent DNA replication system. As spnT over-expression inhibited swarming but not swimming motility, SpnT may indirectly function as a negative regulator of surface-dependent migration and secondary metabolite production.

Bacterial Proteins↗

PubMed2006

Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover.

HD-GYP is a protein domain of unknown biochemical function implicated in bacterial signaling and regulation. In the plant pathogen Xanthomonas campestris pv. campestris, the synthesis of virulence factors and dispersal of biofilms are positively controlled by a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC and by cell-cell signaling mediated by the diffusible signal molecule DSF (diffusible signal factor). The RpfG/RpfC two-component system has been implicated in DSF perception and signal transduction. Here we show that the role of RpfG is to degrade the unusual nucleotide cyclic di-GMP, an activity associated with the HD-GYP domain. Mutation of the conserved H and D residues of the isolated HD-GYP domain resulted in loss of both the enzymatic activity against cyclic di-GMP and the regulatory activity in virulence factor synthesis. Two other protein domains, GGDEF and EAL, are already implicated in the synthesis and degradation respectively of cyclic di-GMP. As with GGDEF and EAL domains, the HD-GYP domain is widely distributed in free-living bacteria and occurs in plant and animal pathogens, as well as beneficial symbionts and organisms associated with a range of environmental niches. Identification of the role of the HD-GYP domain thus increases our understanding of a signaling network whose importance to the lifestyle of diverse bacteria is now emerging.

Amino Acid Sequence↗

PubMed2006

Immunotherapy, immunochemotherapy and chemotherapy for American cutaneous leishmaniasis treatment.

The first choice of treatment for American cutaneous leishmaniasis is the pentavalent antimonial drug. Although it has been shown that this treatment is mostly effective and indicated, some disadvantages should be taken into account such as side effects, long term treatment inconveniences and counter-indication for patients suffering from cardiopathy, nephropathy; yet, aging, pregnancy and other conditions. With the advent of the vaccine anti-American cutaneous leishmaniasis as a prophylactic measure, studies on therapy using the vaccine associated or not with other drugs have been performed by many investigators and it is currently among the alternative treatments and prevention measures for American cutaneous leishmaniasis. In conclusion, the association between antimony and vaccine (immunochemotherapy) showed the same cure rate when compared with the standard treatment (100%) and it was also able to reduce the salt volume in 17.9% and treatment length from 87 to 62 days, decreasing side effects.

Adolescent↗

PubMed2006

Quorum sensing.

Explore the source record for details and available documents.

4-Butyrolactone↗

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

The 1.6A X-ray structure of the unusual c-type cytochrome, cytochrome cL, from the methylotrophic bacterium Methylobacterium extorquens.

The structure of cytochrome cL from Methylobacterium extorquens has been determined by X-ray crystallography to a resolution of 1.6 A. This unusually large, acidic cytochrome is the physiological electron acceptor for the quinoprotein methanol dehydrogenase in the periplasm of methylotrophic bacteria. Its amino acid sequence is completely different from that of other cytochromes but its X-ray structure reveals a core that is typical of class I cytochromes c, having alpha-helices folded into a compact structure enclosing the single haem c prosthetic group and leaving one edge of the haem exposed. The haem is bound through thioether bonds to Cys65 and Cys68, and the fifth ligand to the haem iron is provided by His69. Remarkably, the sixth ligand is provided by His112, and not by Met109, which had been shown to be the sixth ligand in solution. Cytochrome cL is unusual in having a disulphide bridge that tethers the long C-terminal extension to the body of the structure. The crystal structure reveals that, close to the inner haem propionate, there is tightly bound calcium ion that is likely to be involved in stabilization of the redox potential, and that may be important in the flow of electrons from reduced pyrroloquinoline quinone in methanol dehydrogenase to the haem of cytochrome cL. As predicted, both haem propionates are exposed to solvent, accounting for the unusual influence of pH on the redox potential of this cytochrome.

Amino Acid Sequence↗

PubMed2006

A Study for a retrieved implant of ceramic-on-ceramic total hip arthroplasty.

A Mittelmeier-type ceramic on ceramic total hip arthroplasty (THA) was retrieved because of aseptic loosening 17 years after implantation. The extent of wear was assessed by scanning electron microscopy. The synovial fluid and the tissue surrounding the implant were also examined. Scanning electron microscopy analysis of the ball head showed that the main wear zone had grade IV wear. The stripe wear zones showed grain pullout regions (grade 5 wear). Pathologically, chronic inflammation was observed in the surrounding tissue. The debris particles in the synovial fluid were polygonal and approximately 3 x 4 microm. Thus, the Mittelmeier-type THA proved excellent wear resistance. Further longevity of ceramic on ceramic THA may indeed become a reality with improvement of the design and quality of alumina.

Arthroplasty, Replacement, Hip↗

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

Long- and short-chain plant-produced bacterial N-acyl-homoserine lactones become components of phyllosphere, rhizosphere, and soil.

Two N-acyl-homoserine lactone (acyl-HSL) synthase genes, lasI from Pseudomonas aeruginosa and yenI from Yersinia enterocolitica, were introduced into tobacco, individually and in combination. Liquid chromatograph-tandem mass spectrometry and thin-layer chromatography confirmed products of lasI and yenI activity in single and cotransformants. Cotransformants expressing plastid-localized LasI and YenI synthases produced the major acyl-HSLs for each synthase in all tissues tested. Total acyl-HSL signals accumulated in leaf tissue up to 3 pmol/mg of fresh weight, half as much in stem tissue, and approximately 10-fold less in root tissues. Acyl-HSLs were present in aqueous leaf washes from greenhouse-grown transgenic plants. Transgenic lines grown for 14 days under axenic conditions produced detectable levels of acyl-HSLs in root exudates. Ethyl acetate extractions of rhizosphere and nonrhizosphere soil from transgenically grown plants contained active acyl-HSLs, whereas plant-free soil or rhizosphere and nonrhizosphere soil from wild-type plants lacked detectable amounts of acyl-HSLs. This work shows that bioactive acyl-HSLs are exuded from leaves and roots and accumulate in the phytosphere of plants engineered to produce acyl-HSLs. These data further suggest that plants that are bioengineered to synthesize acyl-HSLs can foster beneficial plant-bacteria communications or deter deleterious interactions. Therefore, it is feasible to use bioengineered plants to supplement soils with specific acyl-HSLs to modulate bacterial phenotypes and plant-associated bacterial community structures.

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

A nationwide assessment of the biodiversity value of Uganda's important bird areas network.

BirdLife International's Important Bird Areas (IBA) program is the most developed global system for identifying sites of conservation priority There have been few assessments, however, of the conservation value of IBAs for nonavian taxa. We combined past data with extensive new survey results for Uganda's IBAs in the most comprehensive assessment to date of the wider biodiversity value of a tropical country's IBA network. The combined data set included more than 35,000 site x species records for birds, butterflies, and woody plants at 86 Ugandan sites (23,400 km2), including 29 of the country's 30 IBAs, with data on additional taxa for many sites. Uganda's IBAs contained at least 70% of the country's butterfly and woody plant species, 86% of its dragonflies and 97% of its birds. They also included 21 of Uganda's 22 major vegetation types. For butterflies, dragonflies, and some families of plants assessed, species of high conservation concern were w'ell represented (less so for the latter). The IBAs successfully represented wider biodiversity largely because many have distinctive avifaunas and, as shown by high cross-taxon congruence in complementarity, such sites tended to be distinctive for other groups too. Cross-taxon congruence in overall species richness was weaker and mainly associated with differences in site size. When compared with alternative sets of sites selected using complementarity-based, area-based, or random site-selection algorithms, the IBA network was efficient in terms of the number of sites required to represent species but inefficient in terms of total area. This was mainly because IBA selection considers factors other than area, however which probably improves both the cost-effectiveness of the network and the persistence of represented species.

Animals↗

PubMed2006

N-acylhomoserine lactones antagonize virulence gene expression and quorum sensing in Staphylococcus aureus.

Many gram-negative bacteria employ N-acylhomoserine lactone (AHL)-mediated quorum sensing to control virulence. To determine whether gram-positive bacteria such as Staphylococcus aureus respond to AHLs, we used a growth-dependent lux reporter fusion. Exposure of S. aureus to different AHLs revealed that 3-oxo-substituted AHLs with C10 to C14 acyl chains inhibited light output and growth in a concentration-dependent manner, while short-chain AHLs had no effect. N-(3-Oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) inhibited the production of exotoxins and cell wall fibronectin-binding proteins but enhanced protein A expression. Since these processes are reciprocally regulated via the S. aureus agr quorum-sensing system, which in turn, is regulated via sar, we examined the effect of AHLs on sarA and agr. At sub-growth-inhibitory concentrations of 3-oxo-C12-HSL, both sarA expression and agr expression were inhibited, indicating that the action of 3-oxo-C12-HSL is mediated at least in part through antagonism of quorum sensing in S. aureus. Spent culture supernatants from Pseudomonas aeruginosa, which produces both 3-oxo-C12-HSL and N-butanoyl-homoserine lactone (C4-HSL), also inhibited agr expression, although C4-HSL itself was inactive in this assay. Since quorum sensing in S. aureus depends on the activities of membrane-associated proteins, such as AgrB, AgrC, and AgrD, we investigated whether AHLs perturbed S. aureus membrane functionality by determining their influence on the membrane dipole potential. From the binding curves obtained, a dissociation constant of 7 muM was obtained for 3-oxo-C12-HSL, indicating the presence of a specific saturable receptor, whereas no binding was observed for C4-HSL. These data demonstrate that long-chain 3-oxo-substituted AHLs, such as 3-oxo-C12-HSL, are capable of interacting with the S. aureus cytoplasmic membrane in a saturable, specific manner and at sub-growth-inhibitory concentrations, down-regulating exotoxin production and both sarA and agr expression.

4-Butyrolactone↗

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↗

PubMed2006

Quorum sensing in Yersinia enterocolitica controls swimming and swarming motility.

The Yersinia enterocolitica LuxI homologue YenI directs the synthesis of N-3-(oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL). In a Y. enterocolitica yenI mutant, swimming motility is temporally delayed while swarming motility is abolished. Since both swimming and swarming are flagellum dependent, we purified the flagellin protein from the parent and yenI mutant. Electrophoresis revealed that in contrast to the parent strain, the yenI mutant grown for 17 h at 26 degrees C lacked the 45-kDa flagellin protein FleB. Reverse transcription-PCR indicated that while mutation of yenI had no effect on yenR, flhDC (the motility master regulator) or fliA (the flagellar sigma factor) expression, fleB (the flagellin structural gene) was down-regulated. Since 3-oxo-C6-HSL and C6-HSL did not restore swimming or swarming in the yenI mutant, we reexamined the N-acylhomoserine lactone (AHL) profile of Y. enterocolitica. Using AHL biosensors and mass spectrometry, we identified three additional AHLs synthesized via YenI: N-(3-oxodecanoyl)homoserine lactone, N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL), and N-(3-oxotetradecanoyl)homoserine lactone. However, none of the long-chain AHLs either alone or in combination with the short-chain AHLs restored swarming or swimming in the yenI mutant. By investigating the transport of radiolabeled 3-oxo-C12-HSL and by introducing an AHL biosensor into the yenI mutant we demonstrate that the inability of exogenous AHLs to restore motility to the yenI mutant is not related to a lack of AHL uptake. However, both AHL synthesis and motility were restored by complementation of the yenI mutant with a plasmid-borne copy of yenI.

Bacterial Proteins↗

PubMed2006

A mobile quorum-sensing system in Serratia marcescens.

Quorum-sensing systems that have been widely identified in bacteria play important roles in the regulation of bacterial multicellular behavior by which bacteria sense population density to control various biological functions, including virulence. One characteristic of the luxIR quorum-sensing genes is their diverse and discontinuous distribution among proteobacteria. Here we report that the spnIR quorum-sensing system identified in the enterobacterium Serratia marcescens strain SS-1 is carried in a transposon, TnTIR, which has common characteristics of Tn3 family transposons and is mobile between chromosomes and plasmids of different enterobacterial hosts. SpnIR functions in the new host and was shown to negatively regulate the TnTIR transposition frequency. This finding may help reveal the horizontal transfer and evolutionary mechanism of quorum-sensing genes and alter the way that we perceive regulation of bacterial multicellular behavior.

Chromosomes, Bacterial↗

PubMed2006

Functional analysis of luxS in Staphylococcus aureus reveals a role in metabolism but not quorum sensing.

The function of AI-2 in many bacteria and the physiological role of LuxS, the enzyme responsible for its production, remain matters of debate. Here, we show that in Staphylococcus aureus the luxS gene forms a monocistronic transcriptional unit under the control of a sigma(70)-dependent promoter. The gene was transcribed throughout growth under a variety of conditions, including intracellular growth in MAC-T cells. AI-2 was produced in rich media under aerobic and anaerobic conditions, peaking during the transition to stationary phase, but was hardly detectable in a sulfur-limited defined medium. In the presence of glucose or under anaerobic conditions, cultures retained considerable AI-2 activity after entry into stationary phase. Inactivation of luxS in various S. aureus strains did not affect virulence-associated traits, such as production of hemolysins and extracellular proteases, biofilm formation, and the agr signaling system. Conversely, AI-2 production remained unchanged in an agr mutant. However, luxS mutants grown in a sulfur-limited defined medium exhibited a growth defect. When grown together with the wild type in mixed culture, luxS mutants of various S. aureus strains showed reduced ability to compete for growth under these conditions. In contrast, a complemented luxS mutant grew as well as the parent strain, suggesting that the observed growth defect was of an intracellular nature and had not been caused by either second-site mutations or the lack of a diffusible factor. However, the LuxS/AI-2 system does not appear to contribute to the overall fitness of S. aureus RN6390B during intracellular growth in epithelial cells: the wild type and a luxS mutant showed very similar growth patterns after their internalization by MAC-T cells.

Aerobiosis↗