Paola Cescutti

Exopolysaccharides from Burkholderia cenocepacia inhibit neutrophil chemotaxis and scavenge reactive oxygen species.

Bacteria belonging to the Burkholderia cepacia complex are important opportunistic pathogens in compromised hosts, particularly patients with cystic fibrosis or chronic granulomatous disease. Isolates of B. cepacia complex may produce large amounts of exopolysaccharides (EPS) that endow the bacteria with a mucoid phenotype and appear to facilitate bacterial persistence during infection. We showed that EPS from a clinical B. cenocepacia isolate interfered with the function of human neutrophils in vitro; it inhibited chemotaxis and production of reactive oxygen species (ROS), both essential components of innate neutrophil-mediated host defenses. These inhibitory effects were not due to cytotoxicity or interference with intracellular calcium signaling. EPS also inhibited enzymatic generation of ROS in cell-free systems, indicating that it scavenges these bactericidal products. B. cenocepacia EPS is structurally distinct from Pseudomonas aeruginosa alginate, yet they share the capacity to scavenge ROS and inhibit chemotaxis. These properties could explain why the two bacterial species resist clearance from the infected cystic fibrosis lung.

Structure of the oligomers obtained by enzymatic hydrolysis of the glucomannan produced by the plant Amorphophallus konjac.

Dimers and trimers obtained by enzymatic hydrolysis of the glucomannan produced by the plant Amorphophallus konjac were analysed in order to obtain information on the saccharidic sequences present in the polymer. The polysaccharide was digested with cellulase and beta-mannanase and the oligomers produced were isolated by means of size-exclusion chromatography. They were structurally characterised using electrospray mass spectrometry, capillary electrophoresis, and NMR. The investigation revealed that many possible sequences were present in the polymer backbone suggesting a Bernoulli-type chain.

Interaction of antimicrobial peptides with bacterial polysaccharides from lung pathogens.

The interaction of two cathelicidin antimicrobial peptides, LL-37 and SMAP-29, with three bacterial polysaccharides, respectively, produced by Pseudomonas aeruginosa, Burkholderia cepacia and Klebsiella pneumoniae, was investigated to identify possible mechanisms adopted by lung pathogens to escape the action of innate immunity effectors. In vitro assays indicated that the antibacterial activity of both peptides was inhibited to a variable extent by the three polysaccharides. Circular dichroism experiments showed that these induced an alpha-helical conformation in the two peptides, with the polysaccharides from K. pneumoniae and B. cepacia showing, respectively, the highest and the lowest effect. Fluorescence measurements also indicated the presence of peptide-polysaccharide interactions. A model is proposed in which the binding of peptides to the polysaccharide molecules induces, at low polysaccharide to peptide ratios, a higher order of aggregation, due to peptide-peptide interactions. Overall, these results suggest that binding of the peptides by the polysaccharides produced by lung pathogens can contribute to the impairment of peptide-based innate defenses of airway surface.

Structural determination of the acidic exopolysaccharide produced by a Pseudomonas sp. strain 1.15.

Pseudomonas strain 1.15 was isolated from a freshwater biofilm and shown to produce considerable amounts of an acidic polysaccharide which was investigated by methylation analysis, NMR spectroscopy and ionspray mass spectrometry (ISMS). The polysaccharide was depolymerised by a bacteriophage-associated endoglucosidase and by autohydrolysis, and the resulting oligosaccharides were investigated by NMR spectroscopy and mass spectrometry. The resulting data showed that the parent repeating unit of the 1.15 exopolysaccharide (EPS) is a branched hexasaccharide. The main chain is constituted of the trisaccharide -->4)-alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->3)-beta-D-Glcp- (1--> and the side chain alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->is linked to O-3 of the first Fuc residue. The terminal non-reducing Gal carries a 1-carboxyethylidene acetal in the R configuration at the positions 4 and 6. Of the four different O-acetyl groups present in non-stoichiometric amounts, two were established to be on O-2 of the 3-linked Gal and on O-2 of the 4-linked Fuc.

Macromolecular and solution properties of Cepacian: the exopolysaccharide produced by a strain of Burkholderia cepacia isolated from a cystic fibrosis patient

Light scattering and viscosity measurements were carried out on the previously chemically characterised exopolysaccharide produced by a strain of Burkholderia cepacia isolated from a cystic fibrosis patient. The same exopolysaccharide was also produced by other clinical strains in different laboratories. Therefore, the name Cepacian is now proposed for this exopolysaccharide. Experiments performed as a function of the ionic strength on the native polymer revealed a change in the overall shape of the polymer at low ionic strength. This behaviour was absent in the de-acetylated sample. Potentiometric titrations and light scattering experiments carried out on the acidic form of the native polymer revealed the formation of macromolecular aggregates with a stoichiometry n and 2n stabilised by interactions involving the uronic acid residues.

Activity of antimicrobial peptides in the presence of polysaccharides produced by pulmonary pathogens

Antimicrobial peptides (AMPs) are secreted in the airway and contribute to initial defence against inhaled pathogens. Infections of the respiratory tract are a major cause of morbidity and mortality in preterm newborns and in patients with cystic fibrosis (CF). In this latter group, the state of chronic lung infection is due to the ability of bacteria to grow as mucoid biofilm, a condition characterised by overproduction and release of polysaccharides (PSs). In this study, we investigate the effect of PSs produced by lung pathogens such as Pseudomonas aeruginosa, Klebsiella pneumoniae and members of the Burkholderia cepacia complex on the antibacterial activity of structurally different peptides. The AMPs tested in this study include the cathelicidin LL‐37 and the β‐defensin hBD‐3 from humans, both released at the alveolar level, as well as peptides from other mammals, i.e. SMAP‐29, PG‐1 and Bac7(1‐35). Susceptibility assays, time killing and membrane permeabilization kinetics experiments were carried out to establish whether PSs produced by lung pathogens may be involved in the poor defence reaction of infected lungs and thus explain infection persistence. All the PSs investigated inhibited, albeit to a different extent, the antibacterial activity of the peptides tested, suggesting that their presence in the lungs of patients with CF may contribute to the decreased defence response of this district upon infection by PS‐producing microorganisms. The results also show that inhibition of the antibacterial activity is not simply due to ionic interaction between the negatively charged PSs and the cationic AMPs, but it also involves other structural features of both interactors. Copyright

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 α‐helical conformation interact with exopolysaccharides through electrostatic and other non‐covalent interactions.

Exopolysaccharides produced by a clinical strain of Burkholderia cepacia isolated from a cystic fibrosis patient

Burkholderia cepacia is an opportunistic pathogen involved in pulmonary infections related to cystic fibrosis. A clinical strain, BTS13, was isolated and the production of exopolysaccharides was tested growing the bacteria on two different media, one of which was rich in mannitol as carbon source. The primary structure of the polysaccharides was determined using mostly mass spectrometry and NMR spectroscopy. On both media an exopolysaccharide having the following repeating unit was produced: -->5)-beta-Kdop-(2-->3)-beta-D-Galp2Ac-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-Galp-(1--> This polysaccharide has already been described as the biosynthetic product of another Burkholderia species, B. pseudomallei, the microbial agent causing melioidosis. In addition to this, when grown on the mannitol-rich medium, B. cepacia strain BTS13 produced another polysaccharide that was established to be levan: -->6)-beta-D-Fruf-(2-->. The content of levan was about 20% (w/w) of the total amount of polymers. The ability of B. cepacia to produce these two exopolysaccharides opens new perspectives in the investigation of the role of polysaccharides in lung infections.

Study of the inclusion complexes of aromatic molecules with cyclodextrins using ionspray mass spectrometry

Abstract The formation of inclusion complexes between cyclodextrins (cyclohexa-, cyclohepta-, and cyclooctamylose) and either 1-anilinonaphthalene-8-sulfonate or 2- p -toluidinylnaphthalene-6-sulfonate was investigated by ionspray mass spectrometry operated both in the positive and in the negative ion mode. This soft ionisation technique allowed the detection of the inclusion complexes; the presence of false positives was excluded by increasing the voltage at the orifice which caused breakage of the electrostatic adducts and some fragmentation of the free cyclodextrin molecules, but left the inclusion complexes intact. The spectra recorded in the negative mode showed the presence of complexes formed by two cyclodextrin molecules and one aromatic molecule; such stoichiometry was not detected in the positive mode.

Structural analysis of fructans from Agave americana grown in South Africa for spirit production.

Fructans isolated from Agave americana grown in South Africa are currently used for spirit production. Structural studies on water-soluble fructans were performed to facilitate the development of other applications including its use as a prebiotic. Acid hydrolysis followed by HPAEC-PAD analysis confirmed that the fructan was composed of glucose and fructose, and size analysis by HPAEC-PAD and size exclusion chromatography indicated that the saccharides have a DP range from 6 to 50. An average DP of 14 was estimated by (1)H NMR analysis. Linkage analysis and ESI-MS studies suggest that A. americana has a neofructan structure consisting of a central sucrose to which (2 → 1)- and (2 → 6)-linked β-D-Fruf chains are attached. The (2 → 1)-linked units extend from C-1 of Fru and C-6 of glucose, whereas the (2 → 6)-linked β-D-Fruf units are attached to C-6 of the central Fru. This structure accounts for the presence of equimolar amounts of 1,6-linked Glu and 1,2,6-linked Fru found in linkage analysis and the multiplicity of the NMR signals observed. Detailed ESI-MS studies were performed on fructan fractions: native, periodate oxidized/reduced, and permethylated oligomers. These derivatizations introduced mass differences between Glc and Fru following oxidation and between 1,2-, 1,6-, 2,6-, and 1,2,6-linked units after methylation. Thus, ESI-MS showed the presence of a single Glc per fructan chain and that it is predominantly internal, rather than terminal as found in inulin. These structural features were confirmed by the use of 1D and 2D NMR experiments.