Zugl.: Kiel, Univ., Diss., 2010

Pseudomonas aeruginosa is a common cause of chronic respiratory infection in cystic fibrosis (CF). Infection is established within the lung epithelial mucus layer, through adhesion to mucins. Terminal residues on mucin oligosaccharide chains are highly sulphated and sialylated, which increases their resistance to degradation by bacterial enzymes. However, a number of microbes display mucin sulphatase activity, including P. aeruginosa. Using ion chromatography, the levels of sulphation on different respiratory mucins and the availability of inorganic sulphate in CF sputum were quantified, and the ability of clinical P. aeruginosa isolates to desulphate mucin was tested by providing mucin as a sole sulphur source for growth. All tested P. aeruginosa strains isolated from the CF lung were able to use human respiratory mucin as source of sulphur for growth, whereas other non-clinical Pseudomonas species were not. However, measured levels of inorganic sulphate in CF sputum suggest that bacteria resident in the lung have sufficient inorganic sulphate for growth and are unlikely to require access to mucin-sulphur as a sulphur source during chronic infection. This was confirmed when expression of sulphate-repressed P. aeruginosa genes, atsK and msuE, were found by quantitative PCR to be repressed in CF sputum. These results indicate that sulphate-starvation is unlikely to occur in pathogens residing in CF sputum and, therefore, mucin desulphation may have an alternative purpose in the association between P. aeruginosa and CF airways.Transcriptomic studies showed enhanced expression of 5 main islands on the P. aeruginosa genome in the presence of mucin as a sulphur source, when compared to sulphate. These islands include general sulphur-starvation response gene clusters, encoding desulphurizing enzymes AtsA, SsuD and MsuD, plus the locus PA2083-PA2094. This locus has not been characterised but encodes a putative sulphonatase, an extracellular-function (ECF) type sigma factor, with associated TonB-dependent transducer, and Major Facilitator Superfamily transporters. Transcriptional studies of this locus in response to various sulphur sources revealed that the locus comprises two transcriptional units under sulphate-limited conditions, and putative σ70-type promoters were identified using 5’-RACE and sequence alignment. Transcriptional regulation of the locus is contributed to by the encoded ECF-type σ factor and anti-σ factor, as a mutant carrying only a disrupted copy of these genes displayed a lack of transcriptional downregulation of the locus in the presence of sulphate. The influence of mucin on transcription levels of the locus was also investigated by RT-qPCR, showing that for maximum transcriptional levels both sulphate-limitation and the presence of mucin are required. However, despite repression of P. aeruginosa sulphate-regulated genes in CF sputum, the level of expression of the locus PA2083-PA2094 in CF sputum was comparable to that of P. aeruginosa culture grown in sulphate-limited conditions. The influence of the lung environment may, therefore, have a greater impact on expression levels of the locus than seen in in vitro studies with mucin. To further investigate the role of the locus, mutants were constructed and screened for changes in their ability to utilise a range of sulphur sources, including mucin, for growth. However, none of the mutants showed significant change in their growth patterns in response to any of the other sulphur sources tested, suggesting that the locus may be involved in desulphurization of a compound not tested in this study or may be functionally replaced by other organosulphur utilisation pathways in its absence. With the aim of identifying genes involved in mucin desulphurization, a P. aeruginosa transposon library was generated, combining the high-throughput nature of a random library with the variable expression reporter capabilities afforded by a promoterless GFP insert. The GFP reporter transposon produced varying fluorescence levels over time during growth of individual mutants, based on the activity of the promoter upstream of the transposon insertion site. A preliminary method was devised using fluorescence-activated cell-sorting to isolate mutants displaying altered GFP expression levels in response to sulphate availability and to mucin. Overall, this work explores the prevalence and importance of mucin desulphurization by P. aeruginosa, with relation to cystic fibrosis lungs, and provides some insight into the transcriptional patterns of the P. aeruginosa locus PA2083 to PA2094.

No description available.

No description available.

Background: Excess plugging of small airways is associated with premature death in chronic obstructive pulmonary disease (COPD). Over-expression of beta-epithelial sodium channel (β-ENaC) in airway epithelia in mice resulted in plugging of small airways while cystic fibrosis transmembrane regulator (CFTR) negatively regulated ENaC activity in cell models. Purpose: To test the hypothesis that accumulation of mucus exudates observed with the progression of COPD is related to excess airway epithelial sodium re-absorption as a result of over-expression of β-ENaC and reduced expression of CFTR by small airway epithelia. Methods: Small airway epithelial samples from frozen lungs from patients at different levels of COPD severity were isolated by laser capture microdissection (LCM). β-ENaC, CFTR, and β-actin (control) gene expression was determined by qRT-PCR and compared to expression in entire airways and lung parenchyma surrounding these airways. β-ENaC protein as well as epithelial mucin expression and mucus plugging were localized and quantified after immunohistochemical and periodic acid Schiff staining, respectively. Results: β-ENaC mRNA expression had a strong positive correlation with that of CFTR (p<O.0001) in airway epithelia and surrounding lung parenchyma (p=O.Ol) but not whole airways. β-ENaC mRNA and protein expression were positively correlated (p=O.4O, p=O.O5) and protein expression significantly increased with GOLD stage of COPD severity. Epithelial mucin expression positively correlated with β-ENaC (p=O.38, p=O.O5) and CFTR (p=OAO, p=O.O4.) mRNA and with mucus plugging (p=O. 43 , ptO.OOO2). CFTR mRNA also correlated positively with mucus plugging (p=O. 48 , p=O.O2). Conclusions: Strong positive correlations between β-ENaC and CFTR mRNA expression that are limited to the lung parenchyma and epithelium suggest a novel mechanism of mRNA regulation. This differs from their functional relationship where an inverse relationship between CFTR expression and β-ENaC activity has been reported. Positive correlations of epithelial mucin or mucus plugging with CFTR mRNA but not β-ENaC protein expression in the small airway epithelium suggest that CFTR may regulate mucin at this site independently of β-ENaC protein. The relationship between β-ENaC mRNA andepithelial mucin expression could be due to strong correlations between β-ENaC and CFTR mRNA expression but β-ENaC’s relationship with COPD GOLD stage suggests it may nevertheless play a role in COPD.

Background: Excess plugging of small airways is associated with premature death in chronic obstructive pulmonary disease (COPD). Over-expression of beta-epithelial sodium channel (β-ENaC) in airway epithelia in mice resulted in plugging of small airways while cystic fibrosis transmembrane regulator (CFTR) negatively regulated ENaC activity in cell models. Purpose: To test the hypothesis that accumulation of mucus exudates observed with the progression of COPD is related to excess airway epithelial sodium re-absorption as a result of over-expression of β-ENaC and reduced expression of CFTR by small airway epithelia. Methods: Small airway epithelial samples from frozen lungs from patients at different levels of COPD severity were isolated by laser capture microdissection (LCM). β-ENaC, CFTR, and β-actin (control) gene expression was determined by qRT-PCR and compared to expression in entire airways and lung parenchyma surrounding these airways. β-ENaC protein as well as epithelial mucin expression and mucus plugging were localized and quantified after immunohistochemical and periodic acid Schiff staining, respectively. Results: β-ENaC mRNA expression had a strong positive correlation with that of CFTR (p<O.0001) in airway epithelia and surrounding lung parenchyma (p=O.Ol) but not whole airways. β-ENaC mRNA and protein expression were positively correlated (p=O.4O, p=O.O5) and protein expression significantly increased with GOLD stage of COPD severity. Epithelial mucin expression positively correlated with β-ENaC (p=O.38, p=O.O5) and CFTR (p=OAO, p=O.O4.) mRNA and with mucus plugging (p=O. 43 , ptO.OOO2). CFTR mRNA also correlated positively with mucus plugging (p=O. 48 , p=O.O2). Conclusions: Strong positive correlations between β-ENaC and CFTR mRNA expression that are limited to the lung parenchyma and epithelium suggest a novel mechanism of mRNA regulation. This differs from their functional relationship where an inverse relationship between CFTR expression and β-ENaC activity has been reported. Positive correlations of epithelial mucin or mucus plugging with CFTR mRNA but not β-ENaC protein expression in the small airway epithelium suggest that CFTR may regulate mucin at this site independently of β-ENaC protein. The relationship between β-ENaC mRNA andepithelial mucin expression could be due to strong correlations between β-ENaC and CFTR mRNA expression but β-ENaC’s relationship with COPD GOLD stage suggests it may nevertheless play a role in COPD.

The relationship between human colonic mucosal metabolism and mucin synthesis was explored, with particular reference to ulcerative colitis (UC) and pouchitis. A hypothesis was proposed, that UC and pouchitis result from impaired metabolism of butyrate, and that the outcome of this metabolic event was reduced mucosal protection via effects on mucin synthesis. The study aims were to assess mucosal metabolism in the ileum and colon in controls and in UC, and to assess the effects of agents that are effective therapy for UC on metabolism as measured by mucin synthesis. In histologically normal colonoscopic mucosal biopsies cultured <I>in vitro</I>, the rate of metabolism of butyrate was similar in the ascending (AC) and descending colon (DC). There was a higher rate of metabolism of glutamine in the ascending colon, in agreement with previous work which stressed the relatively greater dependence of the distal colon on butyrate as an energy source. The terminal ileum (TI), in controls had a surprisingly high rate of metabolism of butyrate, significantly higher than the AC, glutamine metabolism in controls was also greater than in the AC. In ulcerative colitis (UC) the most striking change in epithelial metabolism was an increase in the rate of glutamine metabolism in the descending colon. The rates of butyrate metabolism in UC were similar to those in controls, the ratio of butyrate:glutamine metabolism was non-significantly lower in the descending colon in UC as a result of the increased rate of glutamine metabolism. Rates of metabolism in the terminal ileum were similar in UC and controls. Butyrate, at concentrations that are likely to be physiologically and pharmacologically relevant, significantly increased mucin synthesis in colonic mucosal explants from histologically normal and diseased (UC) tissue. Glucocorticoids and nicotine similarly increased colonic mucin synthesis, whereas mineralocorticoids were without effect.

Background: Excess plugging of small airways is associated with premature death in chronic obstructive pulmonary disease (COPD). Over-expression of beta-epithelial sodium channel (β-ENaC) in airway epithelia in mice resulted in plugging of small airways while cystic fibrosis transmembrane regulator (CFTR) negatively regulated ENaC activity in cell models. Purpose: To test the hypothesis that accumulation of mucus exudates observed with the progression of COPD is related to excess airway epithelial sodium re-absorption as a result of over-expression of β-ENaC and reduced expression of CFTR by small airway epithelia. Methods: Small airway epithelial samples from frozen lungs from patients at different levels of COPD severity were isolated by laser capture microdissection (LCM). β-ENaC, CFTR, and β-actin (control) gene expression was determined by qRT-PCR and compared to expression in entire airways and lung parenchyma surrounding these airways. β-ENaC protein as well as epithelial mucin expression and mucus plugging were localized and quantified after immunohistochemical and periodic acid Schiff staining, respectively. Results: β-ENaC mRNA expression had a strong positive correlation with that of CFTR (p<O.0001) in airway epithelia and surrounding lung parenchyma (p=O.Ol) but not whole airways. β-ENaC mRNA and protein expression were positively correlated (p=O.4O, p=O.O5) and protein expression significantly increased with GOLD stage of COPD severity. Epithelial mucin expression positively correlated with β-ENaC (p=O.38, p=O.O5) and CFTR (p=OAO, p=O.O4.) mRNA and with mucus plugging (p=O. 43 , ptO.OOO2). CFTR mRNA also correlated positively with mucus plugging (p=O. 48 , p=O.O2). Conclusions: Strong positive correlations between β-ENaC and CFTR mRNA expression that are limited to the lung parenchyma and epithelium suggest a novel mechanism of mRNA regulation. This differs from their functional relationship where an inverse relationship between CFTR expression and β-ENaC activity has been reported. Positive correlations of epithelial mucin or mucus plugging with CFTR mRNA but not β-ENaC protein expression in the small airway epithelium suggest that CFTR may regulate mucin at this site independently of β-ENaC protein. The relationship between β-ENaC mRNA andepithelial mucin expression could be due to strong correlations between β-ENaC and CFTR mRNA expression but β-ENaC’s relationship with COPD GOLD stage suggests it may nevertheless play a role in COPD. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

The rheological and structural characteristics of gastric (MUC5AC) and duodenal (MUC2) mucin solutions, the structural basis of the adherent mucus layer in the two organs, and their interactions with polyphenols, the phytochemicals which are linked with a number of health benefits, were investigated using particle tracking microrheology and scattering techniques. We used biochemically well characterised porcine mucins as models for human mucins to characterise their viscoelasticity, structure and dynamics as a function of concentration and pH. Additionally, the mesoscopic forces that mediate the integrity of the network were investigated using reducing (dithiothreitol) and chaotropic agents (guanidinium chloride and urea). Mucins in solution were found to be flexible and three distinct viscoelastic regimes were identify ed. At neutral pH, both types of mucin were found to form flexible self-assembled semi-dilute networks above a critical concentration (c*) where the viscosity scales as c 0.53+-0.08 and c 0.53 +-0.06 for MUC5AC and MUC2 respectively. Above a second critical concentration, the entanglement concentration (Ce), the peptide backbones reptate and entangle and there is a sharp increase in viscosity, c 3.92+- 0.38 for MUC5AC and c 5.1 0+-0.08 for MUC2. At low pH, both types of mucin solution undergo a sol-gel transition, forming pH-switchable gels. The addition of tea-derived polyphenols and tea extracts to the mucin solutions has revealed the strong interaction of galloylated phenolic molecules with mucins, which eventually leads to the gelation of the solution. Cross-linking of mucins by galloylated polyphenols is thus expected to have an impact on the physicochemical environment of the stomach and small intestine; the alteration of the organisation of the mucin polymer network is expected to modulate the barrier properties of the two adherent mucus layers which will affect nutrient absorption and the viscoelastic microenvironment of intestinal bacteria.

No description available.