Isolation and characterisation of the biological repeating unit of cepacian, the exopolysaccharide produced by bacteria of the Burkholderia cepacia complex

The repeating unit of cepacian, the exopolysaccharide produced by the majority of the microorganisms belonging to the Burkholderia cepacia complex, was isolated from inner bacterial membranes and investigated by mass spectrometry, with and without prior derivatisation. Interpretation of the mass spectra led to the determination of the biological repeating unit primary structure, thus disclosing the nature of the oligosaccharide produced in vivo. Moreover, mass spectra recorded on the native sample revealed that acetyl substitution was very variable, producing a mixture of repeating units containing zero to four acyl groups. At the same time, finding acetylated oligosaccharides showed that binding of these substituents occurred in the cellular periplasmic space, before the polymerisation process took place. In the chromatographic peak containing the repeating unit, oligosaccharides shorter than the repeating unit co-eluted. Mass spectrometric analysis showed that they were biosynthetic intermediates of the repeating unit and further investigation revealed the biosynthetic sequence of cepacian building block.     

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.     

Exopolysaccharides produced by Inquilinus limosus, a new pathogen of cystic fibrosis patients: novel structures with usual components

The major cause of morbidity and mortality in patients with cystic fibrosis, an autosomal recessive disorder, is chronic microbial colonisation of the major airways that leads to exacerbation of pulmonary infection. Several different microbes colonise cystic fibrosis lungs, and Pseudomonas aeruginosa is one of the most threatening, since the establishment of mucoid (alginate producing) strains is ultimately associated with the patient's death. Very recently a new bacterium, named Inquilinus limosus, was repeatedly found infecting the respiratory tract of cystic fibrosis patients. Its multi-resistance characteristic to antibiotics might result in the spreading of I. limosus infection among the cystic fibrosis community, as recently happened with strains of the Burkholderia cepacia complex. Since exopolysaccharides are recognised as important virulence factors in lung infections, the primary structure of the polysaccharide produced by I. limosus strain LMG 20952(T) was investigated as the first step in understanding its role in pathogenesis. The structure was determined by means of methylation analysis, acid degradations, mass spectrometry and NMR spectroscopy. The results showed that the bacterium produced a mixture constituted of the following polymers: [3)-[4,6-O-(1-carboxyethylidene)]-beta-D-Glcp(1-->]n; [2)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Manp(1-->]n. Both polymers were completely substituted with pyruvyl ketal groups, a novel structural characteristic not previously found in bacterial polysaccharides. The absolute configuration of all pyruvyl groups was S. Inspection of possible local conformations assumed by the two polysaccharide chains showed features, which might provide interesting clues for understanding structure-function relationships.