Inhibition of Pseudomonas aeruginosa ExsA DNA-Binding Activity by N -Hydroxybenzimidazoles

ThePseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence determinant and a potential target for antivirulence drugs. One candidate target is ExsA, a member of the AraC family of DNA-binding proteins required for expression of the T3SS. A previous study identified small molecules based on anN -hydroxybenzimidazole scaffold that inhibit the DNA-binding activity of several AraC proteins, including ExsA. In this study, we further characterized a panel ofN -hydroxybenzimidazoles. The half-maximal inhibitory concentrations (IC50 s) for the testedN -hydroxybenzimidazoles ranged from 8 to 45 μM in DNA-binding assays. Each of theN -hydroxybenzimidazoles protected mammalian cells from T3SS-dependent cytotoxicity, and protection correlated with reduced T3SS gene expression in a coculture infection model. Binding studies with the purified ExsA DNA-binding domain (i.e., lacking the amino-terminal self-association domain) confirmed that the activity ofN -hydroxybenzimidazoles results from interactions with the DNA-binding domain. The interaction is specific, as an unrelated DNA-binding protein (Vfr) was unaffected byN -hydroxybenzimidazoles. ExsA homologs that control T3SS gene expression inYersinia pestis ,Aeromonas hydrophila , andVibrio parahaemolyticus were also sensitive toN -hydroxybenzimidazoles. Although ExsA andY. pestis LcrF share 79% sequence identity in the DNA-binding domain, differential sensitivities to several of theN -hydroxybenzimidazoles were observed. Site-directed mutagenesis based onin silico docking of inhibitors to the DNA-binding domain, and on amino acid differences between ExsA and LcrF, resulted in the identification of several substitutions that altered the sensitivity of ExsA toN -hydroxybenzimidazoles. Development of second-generation compounds targeted to the same binding pocket could lead to drugs with improved pharmacological properties.