Burkholderia cenocepacia is an important respiratory pathogen in persons with cystic fibrosis (CF). Recent studies indicate that B. cenocepacia survives within macrophages and airway epithelial cells in vitro by evading endosomelysosome fusion. We investigated the role of a plasmid-encoded type IV secretion system in the intracellular survival, replication, and processing of B. cenocepacia. Both a wild-type strain (K56-2) and its type IV secretion system mutant (designated LC101) entered and replicated in CF airway epithelial cells and monocyte-derived macrophages. However, significantly more intracellular K56-2 than LC101 bacteria were found in both cell types at 24 h postinfection. Colocalization of bacteria with markers of the classical endocytic pathway indicated that although both K56-2 and LC101 reside transiently in early endosomes, a greater proportion of the mutant bacteria are targeted to lysosomal degradation. In contrast, wild-type bacteria escape from the classical endocytic pathway and traffic to the endoplasmic reticulum, where they replicate. Our results show that the intracellular processing of B. cenocepacia is similar in both professional and nonprofessional phagocytes and that a functional plasmid-encoded type IV secretion system contributes to the survival and replication of B. cenocepacia in eukaryotic cells.

In previous experiments, we have shown that isolates of Pseudomonas cepacia from sputa of patients with cystic fibrosis (CF), particularly those with severe lung infection, exhibited specific binding to purified respiratory or intestinal mucins (U.

Purified rat intestinal mucin was used as a model mucin to study the binding of Escherichia coli serotype 0157:H7, a human pathogen associated with outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. Of six 0157:H7 strains, only one strain (designated CL-49) bound to rat (and other) intestinal mucins by a specific and saturable process. Binding was observed only after the bacteria were serially passaged to promote the expression of type 1 pili (fimbriae). Several other type 1-piliated E. coli strains, however, did not bind to mucin. Binding of E. coli CL-49 was inhibited by D-mannose and short oligomannosyl derivatives, particularly Man-a-1,3-Man, Man-a-1,2-Man, and Man-a-1,3-Man- ,-1,4-N-acetylglucosamine. Other inhibitors of binding included p-nitrophenol (10-4M), heating at 60°C (to remove pili), an antibody to type 1 pili, and purified type 1 pili of E. coli CL-49 used as hapten inhibitors. A comparison of the hydrophobicity of piliated E. coli CL-49 with other type 1-piliated E. coli strains indicated that the former strain was much more hydrophobic than the others. These findings indicate that highly purified intestinal mucins possess specific mannosyl receptor sites for bacterial type 1 pili on E. coli CL-49, but that strong hydrophobic interactions between the mucin and the pili stabilize the mannose-dependent binding process. We speculate that the mucin receptors for type 1 pili reside in oligosaccharides of the 118-kilodalton "link" glycopeptide, since this is the only mucin component known to contain mannose.

Cystic fibrosis (CF) patients develop chronic lung infections associated with airway obstruction by viscous and insoluble mucus secretions. Although mucus glycoproteins (mucins) are thought to be responsible for mucus plugs, other glycoconjugate components of airway secretions have not been systematically evaluated. The aim of the present study was to determine whether chondroitin sulfate proteoglycans (CSPG) contribute to the insolubility of CF sputum. Sputa obtained from 18 CF patients were incubated with chondroitinase ABC (ChABC) or buffer (control) for 18 h at 37°C, and after centrifugation at 12,000 g, the volume of the insoluble pellet and turbidity of the supernatant were determined as measures of solubility. ChABC caused a 70 -90% reduction in supernatant turbidity and a 60 -70% decrease in pellet volume of the 13 purulent CF sputa, but had much less effect on the five nonpurulent CF sputa tested. Similar results were obtained with two non-CF purulent and two non-CF, nonpurulent sputa. Gel electrophoresis, Western blot, and slot blot immunoassays with antichondroitin sulfate and antimucin antibodies revealed that purulent sputa (CF and non-CF) contained more CSPG and less mucin than nonpurulent sputa. In vitro mixing experiments showed that mucin in nonpurulent sputa was reduced upon incubation with purulent sputa, presumably because of degradation or a loss of immunoreactive mucin epitopes from leukocyte and/or bacterial enzymes present in purulent sputa. Our results suggest that CSPG contribute more significantly than mucins to the insolubility of purulent tracheobronchial secretions from CF patients. Because purulent sputa from non-CF patients showed a similar pattern, our observations with CF sputa may have wider applicability. CF is a life-threatening inherited disease caused by genetic mutation of the CF transmembrane conductance regulator (CFTR), a cyclic AMP-dependent chloride channel present in secretory and other cells throughout the body. Despite rapid advances in our knowledge of the structure and function of CFTR, the link between the mutations in this gene and the clinical manifestation of widespread obstruction of exocrine glands with viscous mucus secretions remains obscure.Almost all CF patients develop chronic lung disease related to airway obstruction, inflammation, and recurrent infections that are eventually lethal. In CF airways and intestine, goblet cell hyperplasia and mucin hypersecretion are characteristic, as is the presence of dilated airway submucous glands and intestinal crypt lumina (1-4). Inflammatory cell products stimulate airway secretions and thus contribute to mucus obstruction (4). Secretory mucin macromolecules, consisting of disulfidedependent polymers secreted by epithelial cells, are thought to be largely responsible for the viscoelastic gels and obstructive plugging of bronchial secretions. Another component that contributes to increased viscosity is DNA released from lysed neutrophils in the airways (5,6). This has led to the clinical use of recombinant DNase ...

Purified rat intestinal mucin was used to identify mucin-binding sites for type 1-piliated Escherichia coli 0157:H7 strain CL-49 isolated from a patient with hemorrhagic colitis and hemolytic uremic syndrome. Optimum binding of bacteria in a microtiter binding assay occurred with a mucin coating concentration of 15 ,ug (protein)/150 ,ul. In hapten inhibition studies, several nonmucin glycoproteins bearing exposed mannosyl residues in N-linked oligosaccharides were effective inhibitors, as was rat mucin. The same glycoproteins caused bacterial aggregation. High-molecular-mass glycoproteins of the mucin were separated from its 118-kilodalton "link" glycopeptide fraction, and the latter was shown to be the mucin-binding component for E. coli CL-49 and its purified type 1 pili. This was confirmed in hemagglutination inhibition studies. Treatment of the link glycopeptide with jack bean a-mannosidase or endo-oI-N-acetylglucosaminidase H destroyed bacterial binding activity. Chemical or enzymatic modifications of intact rat mucin were undertaken to evaluate the normal accessibility of the link glycopeptide receptors to E. coli CL-49. Deglycosylation with trifluoromethane-sulfonic acid abolished binding, whereas pronase digestion had no effect. Reduction and alkylation as well as lipid extraction enhanced bacterial binding by the mucin, presumably by causing greater exposure of receptor sites. In summary, our binding studies revealed, for the first time, that intestinal mucin bears oligomannosyl receptors for type 1 pili and that these receptors are located on N-linked oligosaccharides of the 118-kilodalton link glycopeptide region of the mucin. Our experiments suggest the receptors are normally partly "covered" by noncovalently bound lipid. In addition, release of the link component from the rest of the mucin by disulfide bond reduction causes greater exposure of specific bacterium-binding sites.

The carbohydrate composition of winged bean has been studied, The seeds contain about 42.2% total carbohydrates of which starch alone accounts for 36.2%. The total monosaccharides constituted 2.7% and were identified as glucose (1.17%) and fructose (1.5%); oligosaccharides amounted to only 0.61% and were identified as sucrose, raffinose, stachyose, and verbascose, on a dry defatted basis. The cold water soluble gum of winged bean contains glucose and xylose as the major sugars together with a small amount of arabinose while hot water soluble gum was found to be a glucantype polymer. Hemicellulose A consisted of glucose, xylose, and arabinose (15.5:9:1) whereas hemicellulose B consisted exclusively of glucose indicating that it is also a glucan-type polymer.

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The Isolation and physico‐chemical properties of the yam, elephant starch have been investigated. The yield of extracted starch was about 80%. The starch exhibited single stage swelling and moderate solubility in water and low resistance to solubilization by DMSO. The amylose content of the starch was found to be 24.5%. The viscosity decreased considerably during cooking at 90°C. The amylolytic susceptibility of the native and gelatinized starch with human salivary α‐amylase und glucoamylase have also been investigated.