Ruksakiet Kasidid, Stercz Balázs, Tóth Gergő, Jaikumpun Pongsiri, Gróf Ilona, Tengölics Roland, Lohinai Zsolt M, Horváth Péter, Deli Mária A, Steward Martin C, Dobay Orsolya, Zsembery Ákos
Department of Oral Biology, Semmelweis University, H-1089 Budapest, Hungary.
Department of Conservative Dentistry, Semmelweis University, H-1085 Budapest, Hungary.
Biology (Basel). 2021 Jun 10;10(6):519. doi: 10.3390/biology10060519.
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 NaHCO3 in 5% CO2 (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 HCO3− exposure decreased c-di-GMP and increased cAMP levels in a dose-dependent manner. Biofilm formation was also reduced after 48 h exposure to HCO3−. The reciprocal changes in second messenger concentrations were not influenced by changes in medium pH or osmolality. These findings indicate that HCO3− per se modulates the levels of c-di-GMP and cAMP, thereby inhibiting biofilm formation and promoting the planktonic lifestyle of the bacteria.
铜绿假单胞菌生物膜在囊性纤维化（CF）患者体内的形成是导致CF患者发病和死亡的最常见原因之一。环二鸟苷酸（c-di-GMP）和环磷酸腺苷（cAMP）是响应环境信号调节细菌生活方式转变的第二信使。我们旨在研究细胞外pH值和碳酸氢盐对细胞内c-di-GMP和cAMP水平以及生物膜形成的影响。将铜绿假单胞菌接种于脑心浸液培养基中，该培养基在环境空气中分别添加25 mM和50 mM NaCl（pH值分别调至7.4和7.7），或在5%二氧化碳中添加25 mM和50 mM NaHCO₃（pH值为7.4和7.7）。孵育16小时后，提取c-di-GMP和cAMP并测定其浓度。使用实时无标记细胞分析仪和结晶紫测定法研究生物膜的形成。我们的结果表明，暴露于HCO₃⁻会以剂量依赖的方式降低c-di-GMP水平并增加cAMP水平。暴露于HCO₃⁻ 48小时后，生物膜形成也减少。第二信使浓度的反向变化不受培养基pH值或渗透压变化的影响。这些发现表明，HCO₃⁻本身可调节c-di-GMP和cAMP的水平，从而抑制生物膜形成并促进细菌的浮游生活方式。
