Chimeric Ligands of Pili and Lectin A Inhibit Tolerance, Persistence, and Virulence Factors of Pseudomonas aeruginosa over a Wide Range of Phenotypes

[Image: see text] Bacteria readily form resilient phenotypes to counter environmental and antibiotic stresses. Here, we demonstrate a class of small molecules that inhibit a wide range of Pseudomonas aeruginosa phenotypes and enable antibiotics to kill previously tolerant bacteria, preventing the transition of tolerant bacteria into a persistent population. We identified two proteins, type IV pili and lectin LecA, as receptors for our molecules by methods including a new label-free assay based on bacterial motility sensing the chemicals in the environment, the chemical inhibition of bacteriophage adsorption on pili appendages of bacteria, and fluorescence polarization. Structure–activity relationship studies reveal a molecule that inhibits only pili appendage and a class of chimeric ligands that inhibit both LecA and pili. Important structural elements of the ligand are identified for each protein. This selective ligand binding identifies the phenotypes each protein receptor controls. Inhibiting LecA results in reducing biofilm formation, eliminating small colony variants, and is correlated with killing previously tolerant bacteria. Inhibiting pili appendages impedes swarming and twitching motilities and pyocyanin and elastase production. Because these phenotypes are controlled by a broad range of signaling pathways, this approach simultaneously controls the multiple signaling mechanisms preventing bacteria to elude antibiotic treatments.