Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, in which cells are embedded in an extracellular matrix of polymeric molecules. Cell–cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused ion beam–milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell–cell junctions. Combining our in situ observations at cell–cell junctions with biochemistry, native mass spectrometry, and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell–cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell–cell junctions to prevent or treat problematic, chronic bacterial infections.

许多细菌，包括主要的人类病原体铜绿假单胞菌，天然存在于多细胞、耐抗生素的生物膜群落中，其中细胞嵌入聚合物分子的细胞外基质中。铜绿假单胞菌生物膜内的细胞-细胞相互作用由 CdrA 介导，CdrA 是一种存在于生物膜细胞外基质中的大型膜相关粘附素，受环二鸟苷酸的细胞质浓度调节。在这里，使用聚焦离子束研磨标本的电子冷冻断层扫描，我们报告了铜绿假单胞菌细胞外基质中 CdrA 分子的结构完整细胞-细胞连接处的生物膜。将我们在细胞-细胞连接处的原位观察与生物化学、天然质谱和细胞成像相结合，我们证明 CdrA 形成了一个扩展结构，该结构从外膜投射到通过多糖结合伙伴将细胞连接在一起。我们继续使用定制的单域抗体（纳米抗体）结合物来展示 CdrA 的功能重要性。靶向功能性细胞表面 CdrA 分子尖端的纳米抗体可与杀菌抗生素一起用于抑制细菌生物膜形成或破坏先前存在的生物膜。这些结果揭示了细菌生物膜内细胞-细胞相互作用的功能机制，并强调了使用靶向生物膜细胞-细胞连接的抑制剂来预防或治疗问题的前景，