Intercepting second-messenger signaling by rationally designed peptides sequestering c-di-GMP

Significance Cyclic diguanylate (c-di-GMP) regulates a wide range of bacterial cellular functions from biofilm formation to growth and survival. Based on the structural analysis of the complex of c-di-GMP with a bacterial effector protein followed by amino acid sequence optimization, we have developed a short peptide that binds c-di-GMP with nanomolar affinity and high specificity. This provides many opportunities for biotechnological and biomedical applications. In particular, we show that such an endogenously expressed peptide effectively reduces intracellular c-di-GMP and thereby inhibits and even disintegrates biofilms in Pseudomonas aeruginosa.
The bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a wide range of cellular functions from biofilm formation to growth and survival. Targeting a second-messenger network is challenging because the system involves a multitude of components with often overlapping functions. Here, we present a strategy to intercept c-di-GMP signaling pathways by directly targeting the second messenger. For this, we developed a c-di-GMP–sequestering peptide (CSP) that was derived from a CheY-like c-di-GMP effector protein. CSP binds c-di-GMP with submicromolar affinity. The elucidation of the CSP⋅c-di-GMP complex structure by NMR identified a linear c-di-GMP–binding motif, in which a self-intercalated c-di-GMP dimer is tightly bound by a network of H bonds and π-stacking interactions involving arginine and aromatic residues. Structure-based mutagenesis yielded a variant with considerably higher, low-nanomolar affinity, which subsequently was shortened to 19 residues with almost uncompromised affinity. We demonstrate that endogenously expressed CSP intercepts c-di-GMP signaling and effectively inhibits biofilm formation in Pseudomonas aeruginosa, the most widely used model for serious biofilm-associated medical implications.