Abstract
The opportunistic human pathogen Pseudomonas aeruginosa 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 P. aeruginosa 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 P. aeruginosa motility and biofilm formation. In addition, cell culture assays attested a role for PA3177 in the response of P. aeruginosa 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.
Highlights
Pseudomonas aeruginosa is a widespread Gram-negative water and soil bacterium, which is in addition one of the most important opportunistic human pathogens causing severe infections in immunocompromised persons (Hancock and Speert, 2000; Stover et al, 2000)
Further analyses identified the diguanylate cyclase (DGC) PA3177 as part of the HClO-induced stress response and we demonstrate its involvement in motility, biofilm formation and interaction of P. aeruginosa with THP-1 macrophages
Since adhesion of bacterial cells to a surface is among the first steps in the development of biofilms (Hall-Stoodley et al, 2004), we performed static attachment assays in 96-well microtiter plates to show a significant 2.8-fold increase in initial adhesion of P. aeruginosa PAO1 during 2 h exposure to 2 μg/ml NaClO (Figure 1A)
Summary
Pseudomonas aeruginosa is a widespread Gram-negative water and soil bacterium, which is in addition one of the most important opportunistic human pathogens causing severe infections in immunocompromised persons (Hancock and Speert, 2000; Stover et al, 2000). Nucleotide second messengers such as cyclic di-GMP (c-di-GMP), cell-density dependent quorum sensing, sigma factors and two-component regulatory systems play an important role in biofilm formation (Mikkelsen et al, 2011; Fazli et al, 2014) Living within such a biofilm provides bacteria protection against a variety of different stressors like toxic substances, antibiotics or the host immune system and is considered as an adaptive resistance mechanism (Hall-Stoodley et al, 2004; Breidenstein et al, 2011)
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