Structural basis of biofilm formation mediated by the <i>Pseudomonas aeruginosa</i> fibrillar adhesin CdrA

<jats:title>Abstract</jats:title> <jats:p> Many bacteria, including the important human pathogen <jats:italic>Pseudomonas aeruginosa</jats:italic> , are naturally found in antibiotic-tolerant, multicellular biofilms. Cell-cell interactions within <jats:italic>P. aeruginosa</jats:italic> biofilms are mediated by a large fibrillar adhesin called CdrA in an extracellular polysaccharide-dependent manner. Here, we report an electron cryomicroscopy structure of the 60 kDa CdrA adhesive N-terminus, which combined with electron cryotomography of focused-ion beam milled specimens, allows us to derive a complete <jats:italic>in situ</jats:italic> model of the native adhesin. Our structure reveals a small adhesive domain (called ADEPT) at the distal tip of CdrA that is nearly perfectly conserved across the <jats:italic>P. aeruginosa</jats:italic> pangenome, with structural similarity to previously reported sugar-binding domains in multiple bacterial species. Inhibitory nanobodies targeting CdrA that reduce biofilm formation bind to epitopes in, or close to, the ADEPT on bacterial cells. Furthermore, structure-guided mutagenesis of residues within the ADEPT abolishes bacterial aggregation, and genomic deletion of the whole ADEPT leads to strong attenuation of biofilm formation. Our data forms a rational basis for future targeted inhibition of pathogenic <jats:italic>P. aeruginosa</jats:italic> biofilms and elucidates the mechanism of biofilm formation mediated by fibrillar adhesins that are widespread in bacteria. </jats:p>