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.

细菌第二信使环状双鸟苷酸（c-di-GMP）调节从生物膜形成到生长和存活的多种细胞功能。以第二信使网络为目标具有挑战性，因为该系统涉及许多功能经常重叠的组件。在这里，我们提出一种通过直接针对第二个Messenger来拦截c-di-GMP信号通路的策略。为此，我们开发了一种c-di-GMP替代肽（CSP），该肽衍生自CheY样c-di-GMP效应蛋白。CSP以亚微摩尔亲和力结合c-di-GMP。通过NMR阐明CSP⋅c-di-GMP复杂结构，鉴定出线性c-di-GMP结合基序，其中自嵌入的c-di-GMP二聚体通过H键和π网络紧密结合-涉及精氨酸和芳族残基的堆积相互作用。基于结构的诱变产生具有相当高的低纳摩尔亲和力的变体，其随后被缩短为具有几乎不受影响的亲和力的19个残基。我们证明内源性表达的CSP拦截c-di-GMP信号，并有效抑制生物膜的形成铜绿假单胞菌（Pseudomonas aeruginosa），用于严重生物膜相关医学影响的最广泛使用的模型。