Manganese Acts as an Environmental Inhibitor of Pseudomonas aeruginosa Biofilm Development by Inducing Dispersion and Modulating c-di-GMP and Exopolysaccharide Production via RbdA.

The opportunistic human pathogen Pseudomonas aeruginosa causes chronic infections that involve multicellular aggregates called biofilms. Biofilm formation is modulated by the host environment and the presence of cues and/or signals, likely affecting the pool of the bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP). The manganese ion Mn is a divalent metal cation that is essential for pathogenic bacterial survival and replication during the infection in a host organism. In this study, we investigated how Mn alters P. aeruginosa biofilm formation via the regulation of c-di-GMP levels. Exposure to Mn was found to temporally enhance attachment but impair subsequent biofilm development, apparent by reduced biofilm biomass accumulation and lack of microcolony formation due to the induction of dispersion. Moreover, exposure to Mn coincided with reduced production of the exopolysaccharides Psl and Pel, decreased transcriptional abundance of and , and decreased levels of c-di-GMP. To determine whether the effect of Mn was linked to the activation of phosphodiesterases (PDEs), we screened several PDE mutants for Mn-dependent phenotypes (attachment and polysaccharide production) as well as PDE activity. The screen revealed that the PDE RbdA is activated by Mn and is responsible for Mn-dependent attachment, inhibition of Psl production, and dispersion. Taken together, our findings suggest Mn is an environmental inhibitor of P. aeruginosa biofilm development that acts through the PDE RbdA to modulate c-di-GMP levels, thereby impeding polysaccharide production and biofilm formation but enhancing dispersion. While diverse environmental conditions such as the availability of metal ions have been shown to affect biofilm development, little is known about the mechanism. Here, we demonstrate that Mn affects Pseudomonas aeruginosa biofilm development by stimulating phosphodiesterase RbdA activity to reduce the signaling molecule c-di-GMP levels, thereby hindering polysaccharide production and biofilm formation but enhancing dispersion. Our findings demonstrate that Mn acts as an environmental inhibitor of P. aeruginosa biofilms, further suggesting manganese to be a promising new antibiofilm factor.