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Methods in Molecular Biology (Clifton,... 2018We used comparative proteome analysis to determine the target genes of the two quorum sensing (QS) circuits in the opportunistic pathogen Burkholderia cenocepacia: the...
We used comparative proteome analysis to determine the target genes of the two quorum sensing (QS) circuits in the opportunistic pathogen Burkholderia cenocepacia: the N-acyl homoserine lactone (AHL)-based CepIR system and the BDSF (B urkholderia diffusible signal factor, cis-2-dodecenoic acid)-based RpfFR system. In this book chapter, we focus on the description of the practical procedure we currently use in the laboratory to perform a sensitive GeLC-MS/MS shotgun proteomics experiment; we also briefly describe the downstream bioinformatic data analysis.
Topics: Acyl-Butyrolactones; Bacterial Proteins; Burkholderia cenocepacia; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Fatty Acids, Monounsaturated; Intracellular Space; Mass Spectrometry; Peptides; Proteomics; Staining and Labeling; Statistics as Topic
PubMed: 29130174
DOI: 10.1007/978-1-4939-7309-5_15 -
European Journal of Medicinal Chemistry Aug 2017Candida is an important opportunistic human fungal pathogen. The cis-2-dodecenoic acid (BDSF) showing in vitro activity of against C. albicans growth, germ-tube...
Candida is an important opportunistic human fungal pathogen. The cis-2-dodecenoic acid (BDSF) showing in vitro activity of against C. albicans growth, germ-tube germination and biofilm formation has been a potential inhibitor for Candida and other fungi. In this study, facile synthetic strategies toward a novel family of BDSF analogue, 1-alkyl-1H-1,2,3-triazole-4-carboxylic acids (ATCs) was developed. The straightforward synthetic method including converting the commercial available alkyl bromide to alkyl azide, consequently with a typical click chemistry method, copper(II) sulfate and sodium ascorbate as catalyst in water to furnish ATCs with mild to good yields. According to antifungal assay, 1-decyl-4,5-dihydro-1H-1,2,3-triazole-4-carboxylic acid (5d) showed antifungal capability slightly better than BDSF. The 1,2,3-triazole unit played a crucial role for the bioactivity of ATCs was also confirmed when compared with two alkyl-aromatic carboxylic acids. Given its simplicity, high antifungal activity, and wide availability of compounds with halide atoms on the end part of the alkyl chains, the method can be extended to develop more excellent ATC drugs for accomplishing the challenges in future antifungal applications.
Topics: Antifungal Agents; Candida albicans; Click Chemistry; Dose-Response Relationship, Drug; Drug Design; Fatty Acids; Microbial Sensitivity Tests; Molecular Structure; Structure-Activity Relationship; Triazoles
PubMed: 28551587
DOI: 10.1016/j.ejmech.2017.05.001 -
Microbiology (Reading, England) May 2017The opportunistic human pathogen Burkholderia cenocepacia H111 uses two chemically distinct signal molecules for controlling gene expression in a cell density-dependent...
The opportunistic human pathogen Burkholderia cenocepacia H111 uses two chemically distinct signal molecules for controlling gene expression in a cell density-dependent manner: N-acyl-homoserine lactones (AHLs) and cis-2-dodecenoic acid (BDSF). Binding of BDSF to its cognate receptor RpfR lowers the intracellular c-di-GMP level, which in turn leads to differential expression of target genes. In this study we analysed the transcriptional profile of B. cenocepacia H111 upon artificially altering the cellular c-di-GMP level. One hundred and eleven genes were shown to be differentially expressed, 96 of which were downregulated at a high c-di-GMP concentration. Our analysis revealed that the BDSF, AHL and c-di-GMP regulons overlap for the regulation of 24 genes and that a high c-di-GMP level suppresses expression of AHL-regulated genes. Phenotypic analyses confirmed changes in the expression of virulence factors, the production of AHL signal molecules and the biosynthesis of different biofilm matrix components upon altered c-di-GMP levels. We also demonstrate that the intracellular c-di-GMP level determines the virulence of B. cenocepacia to Caenorhabditis elegans and Galleria mellonella.
Topics: Acyl-Butyrolactones; Animals; Burkholderia cenocepacia; Caenorhabditis elegans; Cyclic GMP; Fatty Acids, Monounsaturated; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Moths; Quorum Sensing; Signal Transduction; Virulence; Virulence Factors
PubMed: 28463102
DOI: 10.1099/mic.0.000452 -
Trends in Microbiology Apr 2017The diffusible signaling factor (DSF)-based quorum sensing (QS) system has emerged as a widely conserved cell-cell communication mechanism in Gram-negative bacteria.... (Review)
Review
The diffusible signaling factor (DSF)-based quorum sensing (QS) system has emerged as a widely conserved cell-cell communication mechanism in Gram-negative bacteria. Typically, signals from the DSF family are cis-2-unsaturated fatty acids which regulate diverse biological functions. Recently, substantial progress has been made on the characterization of new members of this family of signals. There have also been new developments in the understanding of the biosynthesis of these molecules where dual enzymatic activities of the DSF synthase and the use of various substrates have been described. The recent discovery of a naturally occurring DSF turnover mechanism and its regulation provides a new dimension in our understanding of how DSF-dependent microorganisms modulate virulence gene expression in response to changes in the surrounding environment.
Topics: Burkholderia cenocepacia; Cronobacter; Fatty Acids; Fatty Acids, Monounsaturated; Gene Expression Regulation, Bacterial; Laurates; Quorum Sensing; Signal Transduction; Virulence; Virulence Factors; Xanthomonas campestris
PubMed: 27979499
DOI: 10.1016/j.tim.2016.11.013 -
Natural Product Research Apr 2017The essential oils obtained by hydrodistillation from aerial parts at different growing stages and root of Cymbocarpum anethoides DC., from Iran were investigated. The...
The essential oils obtained by hydrodistillation from aerial parts at different growing stages and root of Cymbocarpum anethoides DC., from Iran were investigated. The oils were analysed by GC-FID and GC-MS. Forty-five, 52, 40 and 36 components were identified in the essential oils of aerial parts (vegetative, flowering and fruit) and root representing of the 99.2, 99.0, 99.8 and 99.6% of the total oils, respectively. The essential oil of the aerial parts of the plant in vegetative stage was dominated by n-decanal (36.5%) and n-dodecanal (25.3%). n-Decanal (35.8%) and 2E-decenal (25.1%) were the main constituents of the plant oil in flowering stage whereas 2E-decenal (32.1%) and 2E-dodecenal (21.5%) were characterised as the main components of the plant oil in fruit stage. In the essential oil of root, the major identified components were 2-dodecenoic acid (29.8%) and 2E-Dodecenol (12.7%).
Topics: Apiaceae; Fatty Acids, Monounsaturated; Flowers; Fruit; Gas Chromatography-Mass Spectrometry; Iran; Oils, Volatile; Plant Components, Aerial; Plant Oils; Plant Roots
PubMed: 27834096
DOI: 10.1080/14786419.2016.1255884 -
Scientific Reports Sep 2016Xanthomonas campestris pv. campestris (Xcc), a Gram-negative phytopathogenic bacterium, causes black rot disease of cruciferous vegetables. Although Xcc has a complex...
Xanthomonas campestris pv. campestris (Xcc), a Gram-negative phytopathogenic bacterium, causes black rot disease of cruciferous vegetables. Although Xcc has a complex fatty acid profile comprised of straight-chain fatty acids and branched-chain fatty acids (BCFAs), and encodes a complete set of genes required for fatty acid synthesis, there is still little known about the mechanism of BCFA synthesis. We reported that expression of Xcc fabH restores the growth of Ralstonia solanacearum fabH mutant, and this allows the R. solanacearum fabH mutant to produce BCFAs. Using in vitro assays, we demonstrated that Xcc FabH is able to condense branched-chain acyl-CoAs with malonyl-ACP to initiate BCFA synthesis. Moreover, although the fabH gene is essential for growth of Xcc, it can be replaced with Escherichia coli fabH, and Xcc mutants failed to produce BCFAs. These results suggest that Xcc does not have an obligatory requirement for BCFAs. Furthermore, Xcc mutants lost the ability to produce cis-11-methyl-2-dodecenoic acid, a diffusible signal factor (DSF) required for quorum sensing of Xcc, which confirms that the fatty acid synthetic pathway supplies the intermediates for DSF signal biosynthesis. Our study also showed that replacing Xcc fabH with E. coli fabH affected Xcc pathogenesis in host plants.
Topics: Amino Acid Sequence; Bacterial Proteins; Computational Biology; Escherichia coli; Fatty Acids; Quorum Sensing; Sequence Homology, Amino Acid; Signal Transduction; Xanthomonas campestris
PubMed: 27595587
DOI: 10.1038/srep32811 -
Biochemistry Jun 2016Burkholderia cenocepacia is a major concern among respiratory tract infections in cystic fibrosis patients. This pathogen is particularly difficult to treat because of...
Burkholderia cenocepacia is a major concern among respiratory tract infections in cystic fibrosis patients. This pathogen is particularly difficult to treat because of its high level of resistance to the clinically relevant antimicrobial agents. In B. cenocepacia, the quorum sensing cell-cell communication system is involved in different processes that are important for bacterial virulence, such as biofilm formation and protease and siderophore production. Targeting the enzymes involved in this process represents a promising therapeutic approach. With the aim of finding effective quorum sensing inhibitors, we have determined the three-dimensional structure of B. cenocepacia diffusible factor synthase A, DfsA. This bifunctional crotonase (dehydratase/thioesterase) produces the characteristic quorum sensing molecule of B. cenocepacia, cis-2-dodecenoic acid or BDSF, starting from 3-hydroxydodecanoyl-acyl carrier protein. Unexpectedly, the crystal structure revealed the presence of a lipid molecule in the catalytic site of the enzyme, which was identified as dodecanoic acid. Our biochemical characterization shows that DfsA is able to use dodecanoyl-acyl carrier protein as a substrate, demonstrating that dodecanoic acid, the product of this reaction, is released very slowly from the DfsA active site, therefore acting as a DfsA inhibitor. This molecule shows an unprecedented conformational arrangement inside the DfsA active site. In contrast with previous hypotheses, our data illustrate how DfsA and closely related homologous enzymes can recognize long hydrophobic substrates without large conformational changes or assistance by additional regulator molecules. The elucidation of the substrate binding mode in DfsA provides the starting point for structure-based drug discovery studies targeting B. cenocepacia quorum sensing-assisted virulence.
Topics: Amino Acid Sequence; Bacterial Proteins; Burkholderia cenocepacia; Crystallization; Crystallography, X-Ray; Fatty Acids; Gas Chromatography-Mass Spectrometry; Protein Conformation; Quorum Sensing; Sequence Homology, Amino Acid; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity
PubMed: 27198181
DOI: 10.1021/acs.biochem.6b00178 -
Saudi Journal of Biological Sciences May 2016Fatty acid contents of the Peganum harmala plant as a result of hexane extraction were analyzed using GC-MS. The saturated fatty acid composition of the harmal plant was...
Fatty acid contents of the Peganum harmala plant as a result of hexane extraction were analyzed using GC-MS. The saturated fatty acid composition of the harmal plant was tetradecanoic, pentadecanoic, tridecanoic, hexadecanoic, heptadecanoic and octadecanoic acids, while the saturated fatty acid derivatives were 12-methyl tetradecanoic, 5,9,13-trimethyl tetradecanoic and 2-methyl octadecanoic acids. The most abundant fatty acid was hexadecanoic with concentration 48.13% followed by octadecanoic with concentration 13.80%. There are four unsaturated fatty acids called (E)-9-dodecenoic, (Z)-9-hexadecenoic, (Z,Z)-9,12-octadecadienoic and (Z,Z,Z)-9,12,15-octadecatrienoic. The most abundant unsaturated fatty acid was (Z,Z,Z)-9,12,15-octadecatrienoic with concentration 14.79% followed by (Z,Z)-9,12-octadecadienoic with concentration 10.61%. Also, there are eight non-fatty acid compounds 1-octadecene, 6,10,14-trimethyl-2-pentadecanone, (E)-15-heptadecenal, oxacyclohexadecan-2 one, 1,2,2,6,8-pentamethyl-7-oxabicyclo[4.3.1]dec-8-en-10-one, hexadecane-1,2-diol, n-heneicosane and eicosan-3-ol.
PubMed: 27081366
DOI: 10.1016/j.sjbs.2015.04.013 -
Scientific Reports Aug 2015Members of the diffusible signal factor (DSF) family are a novel class of quorum sensing (QS) signals in diverse Gram-negative bacteria. Although previous studies have...
Members of the diffusible signal factor (DSF) family are a novel class of quorum sensing (QS) signals in diverse Gram-negative bacteria. Although previous studies have identified RpfF as a key enzyme for the biosynthesis of DSF family signals, many questions in their biosynthesis remain to be addressed. In this study with the phytopathogen Xanthomonas campestris pv. campestris (Xcc), we show that Xcc produces four DSF-family signals (DSF, BDSF, CDSF and IDSF) during cell culture, and that IDSF is a new functional signal characterized as cis-10-methyl-2-dodecenoic acid. Using a range of defined media, we further demonstrate that Xcc mainly produces BDSF in the presence of carbohydrates; leucine and valine are the primary precursor for DSF biosynthesis; isoleucine is the primary precursor for IDSF biosynthesis. Furthermore, our biochemical analyses show that the key DSF synthase RpfF has both thioesterase and dehydratase activities, and uses 3-hydroxydedecanoyl-ACP as a substrate to produce BDSF. Finally, our results show that the classic fatty acid synthesis elongation cycle is required for the biosynthesis of DSF-family signals. Taken all together, these findings establish a general biosynthetic pathway for the DSF-family quorum sensing signals.
Topics: Amino Acids, Branched-Chain; Bacterial Proteins; Biosynthetic Pathways; Carbohydrates; Cerulenin; Diffusion; Fatty Acids; Genes, Bacterial; Models, Biological; Quorum Sensing; Thiolester Hydrolases; Xanthomonas campestris
PubMed: 26289160
DOI: 10.1038/srep13294 -
Applied and Environmental Microbiology Oct 2015In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within...
In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within the ecological niche. We analyzed the potential interspecies signaling effect of the Burkholderia diffusible signal factor (BDSF) on Francisella novicida, a model organism for Francisella tularensis, and demonstrated that BDSF both inhibits the formation and causes the dispersion of Francisella biofilm. Specificity was demonstrated for the cis versus the trans form of BDSF. Using transcriptome sequencing, quantitative reverse transcription-PCR, and activity assays, we found that BDSF altered the expression of many F. novicida genes, including genes involved in biofilm formation, such as chitinases. Using a chitinase inhibitor, the antibiofilm activity of BDSF was also shown to be chitinase dependent. In addition, BDSF caused an increase in RelA expression and increased levels of (p)ppGpp, leading to decreased biofilm production. These results support our observation that exposure of F. novicida to BDSF causes biofilm dispersal. Furthermore, BDSF upregulated the genes involved in iron acquisition (figABCD), increasing siderophore production. Thus, this study provides evidence for a potential role and mechanism of diffusible signal factor (DSF) signaling in the genus Francisella and suggests the possibility of interspecies signaling between Francisella and other bacteria. Overall, this study suggests that in response to the interspecies DSF signal, F. novicida can alter its gene expression and regulate its biofilm formation.
Topics: Biofilms; Burkholderia; Fatty Acids, Monounsaturated; Francisella tularensis; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Microbial Interactions; Molecular Sequence Data; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; Siderophores
PubMed: 26231649
DOI: 10.1128/AEM.02165-15