Structure of sialyl-oligosaccharides isolated from bronchial mucus glycoproteins of patients (blood group O) suffering from cystic fibrosis

The carbohydrate chains of the bronchial-mucus glycoproteins of six cystic fibrosis patients with blood group O were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high-performance liquid chromatography. Structural characterization was performed by 500-MHz 1H-NMR spectroscopy in combination with quantitative sugar analysis. The established structures range in size from tetra- up to heptasaccharides. They are all sialyl analogs of neutral oligosaccharides that were characterized previously [Lamblin G., Boersma A., Lhermitte M., Roussel P., Mutsaers J. H. G. M., Van Halbeek H. & Vliegenthart J. F. G. (1984) Eur. J. Biochem. 143, 227-236]. The NeuAc residue was found to occur either in alpha (2----3)-linkage to Gal, or in alpha (2----6)-linkage to GalNAc-ol or Gal.     

Human respiratory tract secretions: Mucous glycoproteins of nonpurulent tracheobronchial secretions,and sputum of patients with bronchitis and cystic fibrosis

Mucous glycoproteins were isolated by agarose gel filtration from nonpurulent tracheobronchial secretions and purulent sputum which had been reduced, carboxymethylated and, in the case of purulent secretions, treated with deoxyribonuclease. The solubilized and purified glycoproteins were fractionated on diethylaminoethyl cellulose into two major (I, II) and two minor (Ia, III) blood group active components. Components I and II had similar carbohydrate and amino acid compositions which were typical for human blood group substances. These two components did differ in several respects. Component I contained 1.4–2.6% sulfate and did not inhibit influenza virus hemagglutination while component II contained 7.1–7.8% sulfate and was a potent inhibitor of virus hemagglutination. Component II also migrated more rapidly on sodium dodecyl sulfate-3.3% acrylamide gel electrophoresis. Components I and II in purulent secretions displayed only minor compositional differences from their counterparts in nonpurulent secretions. Component II was more abundant in two sputum samples from subjects with cystic fibrosis than in purulent bronchitic secretions or in nonpurulent secretions.     

Mannosylation of glycoproteins and dolichol derivatives in fibroblasts from patients with cystic fibrosis

Increased incorporation of mannose into endogenous glycoprotein fractions has been found in whole cell lysates and crude membrane preparations of cultured skin fibroblasts from patients with cystic fibrosis (1.3–2.3-times normal) when GDP[¹⁴C]mannose served as the mannosyl donor. In contrast, the incorporation of mannose from GDPmannose into lipid fractions containing dolichol phosphate and dolichol pyrophosphate oligosaccharides as well as the incorporation of mannose from dolichol phospho[³H]mannose into both glycoproteins and dolichol derivatives were not significantly different among cell preparations from patients with cystic fibrosis and normal controls. Mannosyltransferase activity toward exogenous glycoproteins as well as the activities of soluble and membranous α-mannosidase and β-mannosidase appeared to be normal and could not account for the observed differences. The altered incorporation of mannose into endogenous glycoprotein may reflect changes in glycosylation processes other than mannosylation.     

Superantigen types inStaphylococcus aureus isolated from patients with cystic fibrosis

The screening of 17 SAg genes of S. aureus isolated from the sputum of cystic fibrosis (CF) patients revealed that among 47 genetically different strains, 39 (83 %) carried SAg genes. Superantigens forming enterotoxin gene cluster were detected in 20 strains. The 2nd most common superantigen type was selk detected in 13 strains. In 9 strains, selk occurred together with the sea gene. Out of 74 strains recovered from nasal carriers, 56 (75 %) were found to carry SAg genes, 38 carried egc genes, while selk was detected in 5 strains. The predominant SAg types in both investigated S. aureus populations were egc and selk/sea, but selk gene frequency was significantly higher in the CF-derived strains.     

AP-PCR typing of Pseudomonas aeruginosa isolated from patients with cystic fibrosis

Arbitrarily Primed Polymerase Chain Reaction has been used for an epidemiological evaluation of 42 strains of P. aeruginosa isolated from nine cystic fibrosis patients during a three-year investigation period. The resistance patterns of the same strains have also been evaluated. The AP-PCR type fingerprinting was perfomed with primers 10514 and 208. Resistance was evaluated by the Minimal Inhibitory Concentration method. With 10514 eleven different genotypes could be evidenced, while with 208 only five of them could be detected. During the investigation period patients were always colonised by the same genotype. A possible correlation between resistance pattern and genotype with both primers has shown, within the same patient, a correspondence of about 20% for 10514 and a correspondence of only 10% for 208. Patients are colonised by one or two strains of P. aeruginosa and there is no relation between genotype and resistance pattern.     

Matrix metalloprotease-9 dysregulation in lower airway secretions of cystic fibrosis patients

Matrix metalloproteases (MMPs) are proteolytic enzymes that regulate extracellular matrix turnover and aid in restoring tissue architecture following injury. There is an emerging role for extracellular matrix destruction in the pathogenesis of chronic neutrophilic lung diseases. In this study, we examined the expression and activity profiles of MMPs in lower airway secretions from cystic fibrosis (CF) patients, patients with acute respiratory failure (ARF), and normal controls. A discrete repertoire of MMP isoforms was found in the CF samples, with robust MMP-9 expression compared with normal controls and ARF. CF samples possessed increased levels of active MMP-9, as well as decreased amounts of tissue inhibitor of metalloprotease-1 (TIMP-1), a natural inhibitor of MMP-9. The CF inpatient samples demonstrated fully active MMP-9 activity compared with CF outpatients, ARF, and normal controls. CF samples also demonstrated increased human neutrophil elastase (HNE) levels compared with ARF and normal controls. To examine potential mechanisms for the protease dysregulation seen in the CF clinical samples, in vitro studies demonstrated that HNE could activate pro-MMP-9 and also degrade TIMP-1; this HNE-based activation, however, was not seen with MMP-8. A strong correlation was seen between HNE and MMP-9 activity in CF inpatient samples. Finally, the dysregulated MMP-9 activity seen in CF inpatient sputum samples could be significantly reduced by the use of MMP-9 inhibitors. Collectively, these findings further emphasize the proposed protease/antiprotease imbalance in chronic neutrophilic lung disease, providing a potential mechanism contributing to this proteolytic dysregulation.     

Antibacterial and anti-biofilm effects of cathelicidin peptides against pathogens isolated from cystic fibrosis patients

Six different cathelicidin-derived peptides were compared to tobramycin for antibacterial and anti-biofilm effects against S. aureus, P. aeruginosa, and S. maltophilia strains isolated from cystic fibrosis patients. Overall, SMAP-29, BMAP-28, and BMAP-27 showed relevant antibacterial activity (MIC₅₀ 4-8 μg/ml), and in some cases higher than tobramycin. In contrast, indolicidin, LL-37, and Bac7(1-35) showed no significant antimicrobial activity (MIC₅₀ > 32 μg/ml). Killing kinetics experiments showed that in contrast to tobramycin the active cathelicidin peptides exert a rapid bactericidal activity regardless of the species tested. All three peptides significantly reduced biofilm formation by S. maltophilia and P. aeruginosa strains at 1/2× MIC, although at a lower extent than tobramycin. In addition, BMAP-28, as well as tobramycin, was also active against S. aureus biofilm formation. Preformed biofilms were significantly affected by bactericidal SMAP-29, BMAP-27 and BMAP-28 concentrations, although at a lesser extent than tobramycin. Overall, our results indicate the potential of some cathelicidin-derived peptides for the development of novel therapeutic agents for cystic fibrosis lung disease.     

Elastic contributions dominate the viscoelastic properties of sputum from cystic fibrosis patients

Sputum samples from cystic fibrosis (CF) patients were investigated by oscillatory, creep and steady shear rheological techniques over a range of time scales from 10(-3) to 10(6) s. The viscoelastic changes obtained by mixing sputa with the actin-filament-severing protein gelsolin and with the thiol-reducing agent dithiothreitol (DTT) were also investigated. At small strains sputum behaves like a viscoelastic solid rather than a liquid. A nearly constant steady shear viscosity at low shear rates is only observed after long shearing times which cause irreversible changes in the samples. Creep-recovery tests confirm that sputa exhibit viscoelastic properties, with a significant elastic recovery. The results suggest that measurements of elastic moduli, rather than viscosities are more closely related to the mechanical properties of sputum in situ. Severing of actin filaments lowers the elastic modulus by 30-40%, but maintains viscoelastic integrity, while reduction of thiols in the glycoproteins nearly completely fluidizes the samples.     

Genotypic and phenotypic relationships in Burkholderia cepacia isolated from cystic fibrosis patients and the environment

Twenty-one strains of Burkholderia cepacia isolated from the environment, and 21 clinical strains isolated principally from sputum of cystic fibrosis (CF) patients, were characterized genotypically by macrorestriction analysis (genome fingerprinting) and PCR ribotyping, and phenotypically by susceptibility to antibiotics and the ability to macerate onion tissue. The plasmid content of the strains was also investigated. Environmental isolates showed a high degree of genetic variability, all strains differing from both one another and the CF isolates. The CF isolates were less variable, with common strains found in patients attending three geographically distinct CF centres. Phenotypic variation was found both within and between CF and environmental strains. Generally, CF isolates displayed higher levels of antibiotic resistance, while the ability to macerate onion tissue was more prevalent amongst environmental isolates. Plasmids were more frequently found in CF isolates, but were of similar size in both groups of strains. Such variability is not surprising in view of the existence of multiple genomovars within the B. cepacia complex.     

Characterization of Staphylococcus aureus strains isolated from cystic fibrosis patients by conventional and molecular typing

The phenotypic and genotypic characteristics of Staphylococcus aureus strains isolated from respiratory tract of cystic fibrosis (CF) patients were investigated. Slime production, cell-surface hydrophobicity, type of capsular polysaccharide, profile of heteroresistance to methicillin and Sma I restriction profiles were evaluated. S. aureus CF strains have been shown to be heterogeneous in respect to several important features. All of them were slime producing with variation in colony morphology. High or moderate cell-surface hydrophobicity (CSH) was found for, respectively, 16.2% and 83.8% strains. Thirty strains were resistant to methicillin, 60% of them showed heteroresitance and 40% were homoresistant. It was found that 59.6% of strains produced capsular polysaccharides (CP) of 5 or 8 type. Among CP5/CP8 strains, CP8 was the predominant type (81.1%). Typing of 62 CF strains by macrorestriction analysis of chromosomal DNA revealed several major types, differing in their SmaI profiles with a similarity coefficient lower than 0.4. Some of the strains isolated from the same patient at different times of hospitalization, as well as strains isolated at the same time from the relatives, were identical in their PFGE pattern.     

Altered channel properties of porins from Haemophilus influenzae: isolates from cystic fibrosis patients

Changes in amino-acid sequence of the unique pore-forming protein of H. influenzae (OmpP2; porin) have been associated with increased antimicrobial resistance in H. influenzae strains isolated from cystic fibrosis patients. From patients who were subjected to long-term antimicrobial therapy, H. influenzae strains 67d and 69a (patient 27) and strains 77a and 77f (patient 30) were isolated. Strains 67d and 77a were previously shown to have elevated values for minimal inhibitory concentrations of antibiotics compared to strains 69a and 77f. Porins were extracted from all four H. influenzae strains by detergent treatment and purified to homogeneity by ion exchange chromatography. By reconstitution of the clinical Hi porins into planar lipid bilayers, single-channel conductance, ionic selectivity, and voltage-gating characteristics were assessed. Porins 77a and 77f displayed similar single-channel conductance and ionic selectivity. Current-voltage relationships were determined for the different porins: porin 77f displayed substantial voltage gating at both positive and negative polarity; porin 77a gated at negative polarity only. Porins 67d and 69a showed substantial differences in their pore-forming properties: the single-channel conductance of porin 69a was significantly increased (1.05 nS) relative to porin 67d (0.73 nS). Porin 67d was twice as permeable to cations as porin 69a, and at both positive and negative polarities the extent of voltage gating was greater for porin 67d relative to porin 69a. Expression of the porins in an isogenic, porin-deleted H. influenzae background allowed for assessment of the contribution of each porin to the minimum inhibitory concentrations of various antimicrobial compounds. Porin 67d was found to have a decreased susceptibility to the antimicrobials novobiocin and streptomycin. This decreased susceptibility of porin 67d to novobiocin and streptomycin correlates with its decrease in single-channel conductance.     

Chemical and physical properties of human bronchial mucus glycoproteins.

Human bronchial mucus glycoproteins of different chemical types were isolated by Ecteola and gel exclusion chromatography. Chemical analysis indicated polydispersity with regard to content of sulfate and sialic acid. No blood group A, B or H activity was found in these glycoproteins. Compositions are reported for amino acid and sugar residues for several fractions obtained from both cystic fibrotic and chronic bronchitic mucus. It is noteworthy that glycoproteins extracted from a single subject contain molecules with different acid groups as well as significant differences in carbohydrate chain length.     

Interbacterial adhesion between Pseudomonas aeruginosa and indigenous oral bacteria isolated from patients with cystic fibrosis

Interbacterial adhesion between strains of Pseudomonas aeruginosa and strains of indigenous oral bacteria, both of which were isolated from the oral cavity of cystic fibrosis patients, was investigated by the phenomenon of the coaggregation reaction. A total of 22 strains of P. aeruginosa were isolated from the oral cavity of 17 patients and examined for their abilities to coaggregate with 5 strains each of Streptococcus sanguis, Streptococcus mitis, Actinomyces viscosus, and Actinomyces naeslundii. Coaggregation reactions were common between these oral bacteria and both the mucoid and nonmucoid variants of P. aeruginosa. All strains of P. aeruginosa were also able to agglutinate neuraminidase-treated or untreated human erythrocytes of blood types A, B, and O. Positive coaggregation reactions were further characterized by determining the effects of several sugars, and of heat and protease treatments of the bacteria. None of the coaggregtion reactions were inhibited by 0.05 M lactose, galactose, glucose, fucose, or mannose. All coaggregation reactions were dependent upon heat- and protease-sensitive components of the Pseudomonas. Thus, the interbacterial adhesions between P. aeruginosa and the oral bacteria studied appears to involve adhesins on the Pseudomonas cell, which bind to complementary receptors, on the cell surfaces of oral bacteria. The apparent prevalence and diversity of interbacterial adhesions between P. aeruginosa strains originating from the oral cavity of cystic fibrosis patients and strains of the indigenous oral bacteria suggest that some of these reactions may affect the extent to which P. aeruginosa colonizes in the oral cavity of cystic fibrosis patients, and thereby, influence susceptibility of the host to infection.     

Isolation of fatty acids covalently bound to the gastric mucus glycoprotein of normal and cystic fibrosis patients

Covalently bound fatty acids were found in strictly purified and delipidated gastric mucus glycoprotein of normal and cystic fibrosis individuals. The susceptibility of this linkage to methanolic KOH and hydroxylamine treatment indicated the ester bond between fatty acids and glycoprotein. On the average, 2.9 nmol fatty acid/mg glycoprotein were found in normal samples, and 12.2 nmol/mg glycoprotein in samples derived from cystic fibrosis. In normal gastric mucus glycoprotein the covalently linked fatty acids consisted of hexadecanoate (47.0%), octadecanoate (22.0%), tetracosanoate (5.9%), octadecenoate (14.5%) and tetracosenoate (6.0%). In cystic fibrosis mucus glycoprotein the covalently bound fatty acids were comprised mainly of hexadecanoate (36.5%), octadecanoate (48.7%) and octadecenoate (8.6%). These data indicate that cystic fibrosis gastric mucus glycoprotein is highly acylated and perhaps this is the major defect of glycoproteins in this disease.