Bicarbonate Evokes Reciprocal Changes in Intracellular Cyclic di-GMP and Cyclic AMP Levels in Pseudomonas aeruginosa

SIMPLE SUMMARY: Cystic fibrosis (CF) is the most common lethal hereditary disease in Caucasians, causing mainly respiratory and gastrointestinal symptoms. In CF, mutations in the gene encoding an epithelial anion channel cause impaired bicarbonate secretion, which contributes to the formation of thick mucus in the airways. Together with reduced mucociliary clearance, this habitat is ideal for bacterial growth. Biofilm formation contributes to persistent infections and inflammation, as well as higher resistance to antibiotics, and therefore represents one of the major challenges for CF therapy. It is also known that second messenger molecules play pivotal roles in the regulation of bacterial lifestyle. Furthermore, the activities of the enzymes that synthesize or break down these second messengers are sensitive to external pH and bicarbonate. Therefore, we hypothesized that pH and/or bicarbonate could influence bacterial behavior. In this work, we have shown that bicarbonate per se regulates the concentrations of bacterial second messengers and inhibits biofilm formation. These results suggest that bicarbonate could be used as a supportive treatment in CF and possibly in other respiratory diseases associated with chronic bacterial infections and viscous mucus production. ABSTRACT: The formation of Pseudomonas aeruginosa biofilms in cystic fibrosis (CF) is one of the most common causes of morbidity and mortality in CF patients. Cyclic di-GMP and cyclic AMP are second messengers regulating the bacterial lifestyle transition in response to environmental signals. We aimed to investigate the effects of extracellular pH and bicarbonate on intracellular c-di-GMP and cAMP levels, and on biofilm formation. P. aeruginosa was inoculated in a brain–heart infusion medium supplemented with 25 and 50 mM NaCl in ambient air (pH adjusted to 7.4 and 7.7 respectively), or with 25 and 50 mM NaHCO(3) in 5% CO(2) (pH 7.4 and 7.7). After 16 h incubation, c-di-GMP and cAMP were extracted and their concentrations determined. Biofilm formation was investigated using an xCelligence real-time cell analyzer and by crystal violet assay. Our results show that HCO(3)(−) exposure decreased c-di-GMP and increased cAMP levels in a dose-dependent manner. Biofilm formation was also reduced after 48 h exposure to HCO(3)(−). The reciprocal changes in second messenger concentrations were not influenced by changes in medium pH or osmolality. These findings indicate that HCO(3)(−) per se modulates the levels of c-di-GMP and cAMP, thereby inhibiting biofilm formation and promoting the planktonic lifestyle of the bacteria.