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

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Cyclic diguanylate (c-di-GMP) is a crucial bacterial second messenger regulating vital cellular processes.
• Targeting c-di-GMP signaling is complex due to numerous interacting components with overlapping functions.

Purpose of the Study:

• To develop a strategy for intercepting bacterial c-di-GMP signaling by directly targeting the second messenger.
• To create and characterize a novel c-di-GMP-sequestering peptide (CSP) for therapeutic applications.

Main Methods:

• Development of a c-di-GMP-sequestering peptide (CSP) derived from a CheY-like protein.
• Structural elucidation of the CSP·c-di-GMP complex using NMR spectroscopy.
• Structure-based mutagenesis to enhance CSP affinity and shorten the peptide sequence.

Main Results:

• The CSP binds c-di-GMP with submicromolar affinity, with a high-affinity variant achieving low-nanomolar affinity.
• NMR studies revealed a linear binding motif involving specific amino acid interactions.
• Endogenously expressed CSP effectively inhibited biofilm formation in Pseudomonas aeruginosa.

Conclusions:

• A novel, high-affinity c-di-GMP-sequestering peptide (CSP) was successfully developed.
• CSP effectively intercepts c-di-GMP signaling and inhibits biofilm formation in Pseudomonas aeruginosa.
• This strategy offers a promising approach to combat biofilm-associated infections.