Antibiotics elicit a wide range of phenotypic responses from bacteria that cause the bacteria to be more tolerant and resistant to drug treatments. These phenotypes, such as increased biofilm formation or swarming motilities and their associated virulence factors, are correlated to either of two opposite signaling pathways that are governed by high or low levels of global second messenger, bis-(3'-5')-cyclic dimeric guanosine monophosphate (cdG). Here, we show that a class of small organic molecules of disugar-tethered branched hydrocarbons can inhibit all phenotypes of Pseudomonas aeruginosa for the entire range of cdG levels, including biofilm formation, small colony variants, exopolymer production, pellicle formation, swarming motility, elastase, and pyocyanin production. This class of molecules also enables killing of tolerant bacteria and prevents formation of nascent persistent bacteria in biofilms caused by tobramycin treatments. We identify two specific protein receptors, type IV pilin and Lectin A that are targeted by our molecule by multiple methods. This class of chimeric ligand’s binding is validated by a new label-free bacterial motility enabled binding assay that is suitable for identifying new ligand-receptor bindings. We further validated receptor binding by multiple characterization methods including inhibition of pili-specific phage adsorption and delaying phage sensitivity towards P. aeruginosa. Structure-activity studies revealed critical structural elements in the molecule for each protein receptor and correlates the receptor to their controlled phenotypes. Inhibition of LecA protein reduced the cdG level and its phenotypes. While maintaining a low level of cdG by chimeric ligands, inhibition of pili is sufficient to overcome signaling and phenotypes induced by low cdG levels, likely by a mechanism in which the pili appendages are retracted back into the bacteria. Overall, this class of molecules inhibit the virulent phenotypes caused by both high and low cdG levels.

Chimeric ligands of pili and lectin A inhibit drug-tolerance and virulence of pseudomonas aeruginosa over a broad range of second messenger signaling

ACS Fall 2021

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