The limited diversity in targets of available antibiotic therapies has put

tremendous pressure on the treatment of bacterial pathogens, where

numerous resistance mechanisms that counteract their function are becoming

increasingly prevalent. Here, we utilize an unconventional anti-virulence screen

of host-guest interacting macrocycles, and identify a water-soluble synthetic

macrocycle, Pillar[5]arene, that is non-bactericidal/bacteriostatic and has a

mechanism of action that involves binding to both homoserine lactones and

lipopolysaccharides, key virulence factors in Gram-negative pathogens.

Pillar[5]arene is active against Top Priority carbapenem- and third/fourth-

generation cephalosporin-resistant Pseudomonas

aeruginosa and Acinetobacter baumannii, suppressing toxins and biofilms and

increasing the penetration and efficacy of standard-of-care antibiotics in

combined administrations. The binding of homoserine lactones and

lipopolysaccharides also sequesters their direct effects as toxins on eukaryotic

membranes, neutralizing key tools that promote bacterial colonization and

impede immune defenses, both in vitro and in vivo. Pillar[5]arene evades both

existing antibiotic resistance mechanisms, as well as the build-up of rapid

tolerance/resistance. The versatility of macrocyclic host-guest chemistry

provides ample strategies for tailored targeting of virulence in a wide range of

Gram-negative infectious diseases.