The Bep gene cluster in Burkholderia cenocepacia H111 codes for a water-insoluble exopolysaccharide essential for biofilm formation.

Burkholderia cenocepacia is an opportunistic pathogen isolated from cystic fibrosis patients where it causes infections that are extremely difficult to treat with antibiotics, and sometimes have a fatal outcome. Biofilm is a virulence trait of B. cenocepacia, and is associated with infection persistence and increased tolerance to antibiotics. In biofilms exopolysaccharides have an important role, conferring mechanical stability and antibiotic tolerance. Two different exopolysaccharides were isolated from B. cenocepacia H111 biofilms: a water-soluble polysaccharide rich in rhamnose and containing an L-Man residue, and a water-insoluble polymer made of glucose, galactose and mannose. In the present work, the product encoded by B. cenocepacia H111 bepA-L gene cluster was identified as the water-insoluble exopolysaccharide, using mutant strains and NMR spectroscopy of the purified polysaccharides. It was also demonstrated that the B. cenocepacia H111 wild type strain produces the water-insoluble exopolysaccharide in pellicles, thus underlining its potential importance in in vivo infections.

The biofilm of Burkholderia cenocepacia H111 contains an exopolysaccharide composed of l-rhamnose and l-mannose: Structural characterization and molecular modelling.

Burkholderia cenocepacia belongs to the Burkholderia Cepacia Complex, a group of 22 closely related species both of clinical and environmental origin, infecting cystic fibrosis patients. B. cenocepacia accounts for the majority of the clinical isolates, comprising the most virulent and transmissible strains. The capacity to form biofilms is among the many virulence determinants of B. cenocepacia, a characteristic that confers enhanced tolerance to some antibiotics, desiccation, oxidizing agents, and host defenses. Exopolysaccharides are a major component of biofilm matrices, particularly providing mechanical stability to biofilms. Recently, a water-insoluble exopolysaccharide produced by B. cenocepacia H111 in biofilm was characterized. In the present study, a water-soluble exopolysaccharide was extracted from B. cenocepacia H111 biofilm, and its structure was determined by GLC-MS, NMR and ESI-MS. The repeating unit is a linear rhamno-tetrasaccharide with 50% replacement of a 3-α-L-Rha with a α-3-L-Man. [2)-α-L-Rhap-(1→3)-α-L-[Rhap or Manp]-(1→3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→]n Molecular modelling was used to obtain information about local structural motifs which could give information about the polysaccharide conformation.

Proteomic Studies of the Biofilm Matrix including Outer Membrane Vesicles of Burkholderia multivorans C1576, a Strain of Clinical Importance for Cystic Fibrosis.

Biofilms are aggregates of microbial cells encased in a highly hydrated matrix made up of self-produced extracellular polymeric substances (EPS) which consist of polysaccharides, proteins, nucleic acids, and lipids. While biofilm matrix polysaccharides are unraveled, there is still poor knowledge about the identity and function of matrix-associated proteins. With this work, we performed a comprehensive proteomic approach to disclose the identity of proteins associated with the matrix of biofilm-growing Burkholderia multivorans C1576 reference strain, a cystic fibrosis clinical isolate. Transmission electron microscopy showed that B. multivorans C1576 also releases outer membrane vesicles (OMVs) in the biofilm matrix, as already demonstrated for other Gram-negative species. The proteomic analysis revealed that cytoplasmic and membrane-bound proteins are widely represented in the matrix, while OMVs are highly enriched in outer membrane proteins and siderophores. Our data suggest that cell lysis and OMVs production are the most important sources of proteins for the B. multivorans C1576 biofilm matrix. Of note, some of the identified proteins are lytic enzymes, siderophores, and proteins involved in reactive oxygen species (ROS) scavenging. These proteins might help B. multivorans C1576 in host tissue invasion and defense towards immune system assaults.

Burkholderia cenocepacia H111 Produces a Water-Insoluble Exopolysaccharide in Biofilm: Structural Determination and Molecular Modelling.

Biofilms are a multicellular way of life, where bacterial cells are close together and embedded in a hydrated macromolecular matrix which offers a number of advantages to the cells. Extracellular polysaccharides play an important role in matrix setup and maintenance. A water-insoluble polysaccharide was isolated and purified from the biofilm produced by Burkholderia cenocepacia strain H111, a cystic fibrosis pathogen. Its composition and glycosidic linkages were determined using Gas-Liquid Chromatography-Mass Spectrometry (GLC-MS) on appropriate carbohydrate derivatives while its complete structure was unraveled by 1D and 2D NMR spectroscopy in deuterated sodium hydroxide (NaOD) aqueous solutions. All the collected data demonstrated the following repeating unit for the water-insoluble B. cenocepacia biofilm polysaccharide: [3)-α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-d-Galp-(1→3)-α-d-Manp-(1→]n Molecular modelling was used, coupled with NMR Nuclear Overhauser Effect (NOE) data, to obtain information about local structural motifs which could give hints about the polysaccharide insolubility. Both modelling and NMR data pointed at restricted dynamics of local conformations which were ascribed to the presence of inter-residue hydrogen bonds and to steric restrictions. In addition, the good correlation between NOE data and calculated interatomic distances by molecular dynamics simulations validated potential energy functions used for calculations.

Ramachandran conformational energy maps for disaccharide linkages found in Burkholderia multivorans biofilm polysaccharides.

Ramachandran conformational energy maps have been prepared for all of the glycosidic linkages found in the C1576 exopolysaccharide that constitutes the biofilms of the bacterial species Burkholderia multivorans, a member of the Burkholderia cepacian complex that was isolated from a cystic fibrosis patient. This polysaccharide is a rhamnomannan with a tetrasaccharide repeat unit containing two mannose residues and two rhamnose residues, -[3-α-d-Man-(1→2)-α-d-Man-(1→2)-α-d-Rha-(1→3)-α-d-Rha-(1→]n-, where approximately 50% of the rhamnoses are randomly methylated on their O3 hydroxyl groups, further increasing the overall hydrophobicity of the chains. Because of the methylation, the tetrasaccharide repeat unit actually contains six possible linkages. The conformational energy maps are fully adiabatic relaxed maps in which the energy for each (ϕ,ψ) grid point on the map represents the lowest possible energy for the molecule in that conformation, considering all the combinations of the other degrees of freedom, such as hydroxyl orientations. Molecular dynamics simulations were used to verify that these maps indeed describe the conformational dynamics of these linkages. All six linkages were found to be quite restricted in possible ϕ angles, but to exhibit several possible low-energy ψ angles, suggesting that these chains could be quite flexible.

The polysaccharide extracted from the biofilm of Burkholderia multivorans strain C1576 binds hydrophobic species and exhibits a compact 3D-structure.

Microorganisms often grow in communities called biofilms where cells are imbedded in a complex self-produced biopolymeric matrix composed mainly of polysaccharides, proteins, and DNA. This matrix, together with cell proximity, confers many advantages to these microbial communities, but also constitutes a serious concern when biofilms develop in human tissues or on implanted prostheses. Although polysaccharides are considered the main constituents of the matrices, their specific role needs to be clarified. We have investigated the chemical and morphological properties of the polysaccharide extracted from biofilms produced by the C1576 reference strain of the opportunistic pathogen Burkholderia multivorans, which causes lung infections in cystic fibrosis patients. The aim of the present study is the definition of possible interactions of the polysaccharide and the three-dimensional conformation of its chain within the biofilm matrix. Surface plasmon resonance experiments confirmed the ability of the polysaccharide to bind hydrophobic molecules, due to the presence of rhamnose dimers in its primary structure. In addition, atomic force microscopy studies evidenced an extremely compact three-dimensional structure of the polysaccharide which may form aggregates, suggesting a novel view of its structural role into the biofilm matrix.

The spent culture supernatant of Pseudomonas syringae contains azelaic acid.

BACKGROUND: Pseudomonas syringae pv. actinidiae (PSA) is an emerging kiwifruit bacterial pathogen which since 2008 has caused considerable losses. No quorum sensing (QS) signaling molecule has yet been reported from PSA and the aim of this study was to identify possible intercellular signals produced by PSA. RESULTS: A secreted metabolome analysis resulted in the identification of 83 putative compounds, one of them was the nine carbon saturated dicarboxylic acid called azelaic acid. Azelaic acid, which is a nine-carbon (C9) saturated dicarboxylic acid, has been reported in plants as a mobile signal that primes systemic defenses. In addition, its structure,(which is associated with fatty acid biosynthesis) is similar to other known bacterial QS signals like the Diffusible Signal Facor (DSF). For these reason it could be acting as s signal molecule. Analytical and structural studies by NMR spectroscopy confirmed that in PSA spent supernatants azelaic acid was present. Quantification studies further revealed that 20 µg/L of were present and was also found in the spent supernatants of several other P. syringae pathovars. The RNAseq transcriptome study however did not determine whether azelaic acid could behave as a QS molecule. CONCLUSIONS: This study reports of the possible natural biosynthesis of azelaic acid by bacteria. The production of azelaic acid by P. syringae pathovars can be associated with plant-bacteria signaling.

Tramesan, a novel polysaccharide from Trametes versicolor. Structural characterization and biological effects.

Mushrooms represent a formidable source of bioactive compounds. Some of these may be considered as biological response modifiers; these include compounds with a specific biological function: antibiotics (e.g. plectasin), immune system stimulator (e,g, lentinan), antitumor agents (e.g. krestin, PSK) and hypolipidemic agents (e.g. lovastatin) inter alia. In this study, we focused on the Chinese medicinal mushroom "yun zhi", Trametes versicolor, traditionally used for (cit.) "replenish essence and qi (vital energy)". Previous studies indicated the potential activity of extracts from culture filtrate of asexual mycelia of T. versicolor in controlling the growth and secondary metabolism (e.g. mycotoxins) of plant pathogenic fungi. The quest of active principles produced by T. versicolor, allowed us characterising an exo-polysaccharide released in its culture filtrate and naming it Tramesan. Herein we evaluate the biological activity of Tramesan in different organisms: plants, mammals and plant pathogenic fungi. We suggest that the bioactivity of Tramesan relies mostly on its ability to act as pro antioxidant molecule regardless the biological system on which it was applied.

Biofilms produced by Burkholderia cenocepacia: influence of media and solid supports on composition of matrix exopolysaccharides.

Bacteria usually grow forming biofilms, which are communities of cells embedded in a self-produced dynamic polymeric matrix, characterized by a complex three-dimensional structure. The matrix holds cells together and above a surface, and eventually releases them, resulting in colonization of other surfaces. Although exopolysaccharides (EPOLs) are important components of the matrix, determination of their structure is usually performed on samples produced in non-biofilm conditions, or indirectly through genetic studies. Among the Burkholderia cepacia complex species, Burkholderia cenocepacia is an important pathogen in cystic fibrosis (CF) patients and is generally more aggressive than other species. In the present investigation, B. cenocepacia strain BTS2, a CF isolate, was grown in biofilm mode on glass slides and cellulose membranes, using five growth media, one of which mimics the nutritional content of CF sputum. The structure of the matrix EPOLs was determined by 1H-NMR spectroscopy, while visualization of the biofilms on glass slides was obtained by means of confocal laser microscopy, phase-contrast microscopy and atomic force microscopy. The results confirmed that the type of EPOLs biosynthesized depends both on the medium used and on the type of support, and showed that mucoid conditions do not always lead to significant biofilm production, while bacteria in a non-mucoid state can still form biofilm containing EPOLs.

A novel rhamno-mannan exopolysaccharide isolated from biofilms of Burkholderia multivorans C1576.

Burkholderia multivorans C1576 is a Gram negative opportunistic pathogen causing serious lung infection in cystic fibrosis patients. Considering that bacteria naturally form biofilms, and exopolysaccharides are recognized as important factors for biofilm architecture set-up, B. multivorans was grown both in biofilm and in non-biofilm mode on two different media in order to compare the exopolysaccharides biosynthesized in these different experimental conditions. The exopolysaccharides produced were purified and their structure was determined resorting mainly to NMR spectroscopy, ESI mass spectrometry and gas chromatography coupled to mass spectrometry. The experimental data showed that both in biofilm and non-biofilm mode B. multivorans C1576 produced a novel exopolysaccharide having the following structure: [Formula: see text]. About 50% of the 2-linked rhamnose residues are substituted on C-3 with a methyl ether group. The high percentage of deoxysugar Rha units, coupled with OMe substitutions, suggest a possible role for polymer domains with marked hydrophobic characteristics able to create exopolysaccharide junction zones favouring the stability of the biofilm matrix.

Versatility of the Burkholderia cepacia complex for the biosynthesis of exopolysaccharides: a comparative structural investigation.

The Burkholderia cepacia Complex assembles at least eighteen closely related species that are ubiquitous in nature. Some isolates show beneficial potential for biocontrol, bioremediation and plant growth promotion. On the contrary, other strains are pathogens for plants and immunocompromised individuals, like cystic fibrosis patients. In these subjects, they can cause respiratory tract infections sometimes characterised by fatal outcome. Most of the Burkholderia cepacia Complex species are mucoid when grown on a mannitol rich medium and they also form biofilms, two related characteristics, since polysaccharides are important component of biofilm matrices. Moreover, polysaccharides contribute to bacterial survival in a hostile environment by inhibiting both neutrophils chemotaxis and antimicrobial peptides activity, and by scavenging reactive oxygen species. The ability of these microorganisms to produce exopolysaccharides with different structures is testified by numerous articles in the literature. However, little is known about the type of polysaccharides produced in biofilms and their relationship with those obtained in non-biofilm conditions. The aim of this study was to define the type of exopolysaccharides produced by nine species of the Burkholderia cepacia Complex. Two isolates were then selected to compare the polysaccharides produced on agar plates with those formed in biofilms developed on cellulose membranes. The investigation was conducted using NMR spectroscopy, high performance size exclusion chromatography, and gas chromatography coupled to mass spectrometry. The results showed that the Complex is capable of producing a variety of exopolysaccharides, most often in mixture, and that the most common exopolysaccharide is always cepacian. In addition, two novel polysaccharide structures were determined: one composed of mannose and rhamnose and another containing galactose and glucuronic acid. Comparison of exopolysaccharides obtained from cultures on agar plates with those extracted from biofilms on cellulose membranes showed important differences, thus suggesting that extrapolating data from non-biofilm conditions might not always be applicable.

Structure of a novel exopolysaccharide produced by Burkholderia vietnamiensis, a cystic fibrosis opportunistic pathogen.

Burkholderia vietnamiensis belongs to the Burkholderia cepacia complex and is an opportunistic pathogen for cystic fibrosis patients. As many other Burkholderia species, it has a mucoide phenotype, producing abundant exopolysaccharide. In general, polysaccharides contribute to bacterial survival in a hostile environment, are recognised as virulence factors and as important components in biofilm formation. The primary structure of the exopolysaccharide produced by B. vietnamiensis LMG 10929 was determined mainly by use of 1D and 2D NMR spectroscopy and ESI mass spectrometry. The polymer consists of the trisaccharidic backbone 3)-ß-D-Glcp-(1→4)-α-D-Glcp-(1→3)-α-L-Fucp-(1→ with the side chain α-D-Glcp-(1→4)-α-D-GlcAp-(1→3)-α-L-Fucp-(1→ linked to C-3 of the α-D-Glcp residue. The polysaccharide also bears acetyl substituents on about 20% of its repeating units and on at least two different positions. The presence of fucose residues is a novel structural feature among the exopolysaccharides produced by species of the B. cepacia complex.

Conformational properties of two exopolysaccharides produced by Inquilinus limosus, a cystic fibrosis lung pathogen.

Inquilinus limosus is a multi-resistant bacterium found in the respiratory tract of patients with cystic fibrosis. This bacterium produces two unique fully pyruvylated exopolysaccharides in similar quantities: an α-(1→2)-linked mannan and a ß-(1→3)-linked glucan. We employed molecular modelling methods to probe the characteristic conformations and dynamics of these polysaccharides, with corroboration from potentiometric titrations and circular dichroism experiments. Our calculations reveal different structural motifs for the mannan and glucan polysaccharides: the glucan forms primarily right-handed helices with a wide range of extensions, while the mannan forms only left-handed helices. This finding is supported by our circular dichroism experiments. Our calculations also show that the (1→3)-ß-d-Glcp linkage is more dynamically flexible than the (1→2)-α-d-Manp: the glucan characteristically forms a range of wide helices with large central cavities. In contrast, the mannan forms rigid regular 'bottlebrush' helices with a minimal central cavity. The widely different character of these two polymers suggests a possible differentiation of biological roles.

Investigation of bacterial resistance to the immune system response: cepacian depolymerisation by reactive oxygen species.

Reactive oxygen species (ROS) are part of the weapons used by the immune system to kill and degrade infecting microorganisms. Bacteria can produce macromolecules, such as polysaccharides, that are able to scavenge ROS. Species belonging to the Burkholderia cepacia complex are involved in serious lung infection in cystic fibrosis patients and produce a characteristic polysaccharide, cepacian. The interaction between ROS and bacterial polysaccharides was first investigated by killing experiments, where bacteria cells were incubated with sodium hypochlorite (NaClO) with and without prior incubation with cepacian. The results showed that the polysaccharide had a protective effect towards bacterial cells. Cepacian was then treated with different concentrations of NaClO and the course of reactions was followed by means of capillary viscometry. The degradation products were characterised by size-exclusion chromatography, NMR and mass spectrometry. The results showed that hypochlorite depolymerised cepacian, removed side chains and O-acetyl groups, but did not cleave the glycosidic bond between glucuronic acid and rhamnose. The structure of some oligomers produced by NaClO oxidation is reported.

O-Acetyl location on cepacian, the principal exopolysaccharide of Burkholderia cepacia complex bacteria.

Cepacian is an exopolysaccharide produced by the majority of the isolates belonging to the Burkholderia cepacia complex bacteria, a group of 17 species, some of which infect cystic fibrosis patients, sometime with fatal outcome. The repeating unit of cepacian consists of a backbone having a trisaccharidic repeating unit with three side chains, as reported in the formula below. The exopolysaccharide is also acetylated, carrying from one to three acetyl esters per repeating unit, depending on the strain examined. The consequences of O-acetyl substitution in a polysaccharide are important both for its biological functions and for industrial applications, including the preparation of conjugated vaccines, since O-acetyl groups are important immunogenic determinants. The location of acetyl groups was achieved by NMR spectroscopy and ESI mass spectrometry and revealed that these substituents are scattered in non-stoichiometric ratio on many sugar residues in different positions, a feature which adds to the already unique carbohydrate structure of the polysaccharide.

A novel highly charged exopolysaccharide produced by two strains of Stenotrophomonas maltophilia recovered from patients with cystic fibrosis.

Stenotrophomonas maltophilia is a non-fermenting Gram-negative microorganism capable of causing chronic pulmonary infection in cystic fibrosis patients and its ability to form biofilms on polystyrene and glass surfaces, as well as on cystic fibrosis-derived bronchial epithelial IB3-I cells was recently demonstrated. The latter evidence might explain the power of S. maltophilia to produce persistent lung infections, despite intensive antibiotic treatment. In addition to being important components of the extracellular biofilm matrix, polysaccharides are involved in virulence, as they contribute to bacterial survival in a hostile environment. With the aim of contributing to the elucidation of S. maltophilia virulence factors, the exopolysaccharides produced by two mucoid clinical isolates of S. maltophilia obtained from two cystic fibrosis patients were completely characterised, mainly by means of ESI-MS and NMR spectroscopy. The results showed that, although the two isolates were recovered from two different patients living in different countries (Italy and France), the exopolysaccharides produced have an identical primary structure, with the following repeating unit: The exopolysaccharide is highly negatively charged for the presence of three uronic acids on four residues in the repeating unit. Moreover, an ether-linked d-lactate substituent is located on C-3 and one O-acetyl group on C-4 of the galacturonic acid side chain. Another O-acetyl group substitutes C-2 of the galacturonic acid in the backbone, making this primary structure unique.

The lipid A of Burkholderia multivorans C1576 smooth-type lipopolysaccharide and its pro-inflammatory activity in a cystic fibrosis airways model.

Cystic fibrosis is an autosomal recessive disorder and it is characterised by chronic bacterial airway infection which leads to progressive lung deterioration, sometimes with fatal outcome. Burkholderia multivorans and Burkholderia cenocepacia are the species responsible for most of the infections of cystic fibrosis patients. Lipopolysaccharide endotoxins (LPSs) are among the foremost factors of pathogenesis of Gram-negative infection and, in particular, lipid A is the endotoxic portion of LPS responsible for eliciting host innate immune response. In this work, the complete primary structure of the lipid A from B. multivorans C1576 has been defined and, further, its pro-inflammatory activity in a cystic fibrosis airways model is shown. The structure of B. multivorans lipid A was attained by chemical, mass spectrometry and nuclear magnetic resonance analyses whereas its biological activity was assessed on the intestinal epithelial cell line CACO-2 cells, on the airway epithelial IB3-1 cells, carrying the ΔF508/W1282X CFTR mutation and on an ex vivo model of culture explants of nasal polyps.

Structural study and conformational behavior of the two different lipopolysaccharide O-antigens produced by the cystic fibrosis pathogen Burkholderia multivorans.

Lipopolysaccharides (LPSs) are virulence factors expressed by gram-negative bacteria; they are among those mainly responsible for bacterial virulence. In this work we define the primary structure and the conformational features of the O-chain from the LPS produced by the highly virulent clinical isolate Burkholderia multivorans strain C1576, an opportunistic human pathogen isolated in a cystic fibrosis center and causative of an outbreak with lethal outcome. We demonstrate that the LPS from this clinical isolate consists of two O-polysaccharide chains present in different amounts and made up of repeating units, both containing deoxy sugar. Additionally, conformational studies have been performed to establish and compare the spatial arrangements of the two polysaccharides and differences in their shape have been highlighted. The comprehension of the structural and conformational features of the two repeating units may help to explain their biological significance, the molecular shape of the bacterial external surface, and the comprehension at the molecular level of the recognition mechanisms of the antibodies.

Inhibition of cathelicidin activity by bacterial exopolysaccharides.

The interaction of bacterial exopolysaccharides, produced by opportunistic lung pathogens, with antimicrobial peptides of the innate primate immune system was investigated. The exopolysaccharides were produced by Pseudomonas aeruginosa, Inquilinus limosus and clinical isolates of the Burkholderia cepacia complex, bacteria that are all involved in lung infections of cystic fibrosis patients. The effects of the biological activities of three orthologous cathelicidins from Homo sapiens sapiens, Pongo pygmaeus (orangutan) and Presbitys obscurus (dusky leaf monkey) were examined. Inhibition of the antimicrobial activity of peptides was assessed using minimum inhibitory concentration assays on a reference Escherichia coli strain in the presence and absence of exopolysaccharides, whereas complex formation between peptides and exopolysaccharides was investigated by means of circular dichroism, fluorescence spectroscopy and atomic force microscopy. Biological assays revealed that the higher the negative charge of exopolysaccharides the stronger was their inhibiting effect. Spectroscopic studies indicated the formation of molecular complexes of varying stability between peptides and exopolysaccharides, explaining the inhibition. Atomic force microscopy provided a direct visualization of the molecular complexes. A model is proposed where peptides with an alpha-helical conformation interact with exopolysaccharides through electrostatic and other non-covalent interactions.

Macromolecular properties of cepacian in water and in dimethylsulfoxide.

Cepacian is the exopolysaccharide produced by the majority of the so far investigated clinical strains of the Burkholderia cepacia complex. This is a group of nine closely related bacterial species that might cause serious lung infections in cystic fibrosis patients, in some cases leading to death. In this paper the aggregation ability and the conformational properties of cepacian chain were investigated to understand its role in biofilm formation. Viscosity and atomic force microscopy studies in water and in mixed (dimethylsulfoxide/water) solvent indicated the formation of double stranded molecular structures in aqueous solutions. Inter-residue short distances along cepacian chain were investigated by NOE NMR, which showed that two side chains of cepacian were not conformationally free due to strong interactions with the polymer backbone. These interactions were attributed to hydrogen bonding and contributed to structure rigidity.