PURPOSE: To measure the effect of extracellular matrix substrate, pH, and O(2) on Pseudomonas aeruginosa binding. METHODS: Extracellular matrix substrates were prepared from human corneal epithelial cells cultured in 2% or 20% O(2). P. aeruginosa strains ATCC 19660 or PAO1 (suspended in pH 7.0 or 7.5 buffer) were cultured on extracellular matrix substrates in 2% or 20% O(2). The mean number of adherent bacteria per counted per field +/- SEM (n = 15) was determined for combinations of bacteria, extracellular matrix substrate, pH, and O(2). Binding in the presence of antibodies directed against laminin-5 was also measured. RESULTS: Extracellular matrix substrates produced by cells cultured in 20% O(2), combined with an environment of pH 7.0, provided the least favorable conditions for binding of strain 19660. In contrast, extracellular matrix substrates produced by cells cultured in 2% O(2), combined with an environment of pH 7.0, provided the most favorable conditions for binding of strain 19660. Binding of PAO1, however, as a function of extracellular matrix substrate and pH, did not similarly compare with binding of strain 19660. Antibodies against laminin-5 chains served to increase the number of strain 19660 bacteria bound to extracellular matrix substrates compared with the control. CONCLUSIONS: The extracellular matrix secreted by hypoxic corneal epithelial cells is a substrate for binding of P. aeruginosa. Results in previous studies have shown that hypoxic extracellular matrix contains less laminin-5 protein than normoxic matrix. The antibody studies in this report suggest that the decrease in laminin-5 content in hypoxic matrix, relative to matrix secreted by normoxic corneal epithelium, may be responsible for increased bacterial adhesion.
PURPOSE: To measure the effect of extracellular matrix substrate, pH, and O(2) on Pseudomonas aeruginosa binding. METHODS: Extracellular matrix substrates were prepared from human corneal epithelial cells cultured in 2% or 20% O(2). P. aeruginosa strains ATCC 19660 or PAO1 (suspended in pH 7.0 or 7.5 buffer) were cultured on extracellular matrix substrates in 2% or 20% O(2). The mean number of adherent bacteria per counted per field +/- SEM (n = 15) was determined for combinations of bacteria, extracellular matrix substrate, pH, and O(2). Binding in the presence of antibodies directed against laminin-5 was also measured. RESULTS: Extracellular matrix substrates produced by cells cultured in 20% O(2), combined with an environment of pH 7.0, provided the least favorable conditions for binding of strain 19660. In contrast, extracellular matrix substrates produced by cells cultured in 2% O(2), combined with an environment of pH 7.0, provided the most favorable conditions for binding of strain 19660. Binding of PAO1, however, as a function of extracellular matrix substrate and pH, did not similarly compare with binding of strain 19660. Antibodies against laminin-5 chains served to increase the number of strain 19660 bacteria bound to extracellular matrix substrates compared with the control. CONCLUSIONS: The extracellular matrix secreted by hypoxic corneal epithelial cells is a substrate for binding of P. aeruginosa. Results in previous studies have shown that hypoxic extracellular matrix contains less laminin-5 protein than normoxic matrix. The antibody studies in this report suggest that the decrease in laminin-5 content in hypoxic matrix, relative to matrix secreted by normoxic corneal epithelium, may be responsible for increased bacterial adhesion.