The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa

Abstract

The opportunistic human pathogen <i>Pseudomonas aeruginosa</i> is able to survive under a variety of often harmful environmental conditions due to a multitude of intrinsic and adaptive resistance mechanisms, including biofilm formation as one important survival strategy. Here, we investigated the adaptation of <i>P. aeruginosa</i> PAO1 to hypochlorite (HClO), a phagocyte-derived host defense compound and frequently used disinfectant. In static biofilm assays, we observed a significant enhancement in initial cell attachment in the presence of sublethal HClO concentrations. Subsequent LC-MS analyses revealed a strong increase in cyclic-di-GMP (c-di-GMP) levels suggesting a key role of this second messenger in HClO-induced biofilm development. Using DNA microarrays, we identified a 26-fold upregulation of ORF PA3177 coding for a putative diguanylate cyclase (DGC), which catalyzes the synthesis of the second messenger c-di-GMP - an important regulator of bacterial motility, sessility and persistence. This DGC PA3177 was further characterized in more detail demonstrating its impact on <i>P. aeruginosa</i> motility and biofilm formation. In addition, cell culture assays attested a role for PA3177 in the response of <i>P. aeruginosa</i> to human phagocytes. Using a subset of different mutants, we were able to show that both Pel and Psl exopolysaccharides are effectors in the PA3177-dependent c-di-GMP network.