The PqsE active site as a target for small molecule antimicrobial agents againstPseudomonas aeruginosa

The opportunistic pathogenPseudomonas aeruginosacauses antibiotic resistant, nosocomial infections in immuno-compromised individuals, and is a high priority for antimicrobial development. Key to pathogenicity inP. aeruginosaare biofilm formation and virulence factor production. Both traits are controlled by the cell-to-cell communication process called quorum sensing (QS). QS involves the synthesis, release, and population-wide detection of signal molecules called autoinducers. We previously reported that activity of the RhlR QS transcription factor depends on a protein-protein interaction with the hydrolase, PqsE, and PqsE catalytic activity is dispensable for this interaction. Nonetheless, the PqsE-RhlR interaction could be disrupted by substitution of an active site glutamate residue with tryptophan (PqsE(E182W)). Here, we show that disruption of the PqsE-RhlR interaction via either the E182W change or alteration of PqsE surface residues that are essential for the interaction with RhlR, attenuatesP. aeruginosainfection in a murine host. We use crystallography to characterize the conformational changes induced by the PqsE(E182W) substitution to define the mechanism underlying disruption of the PqsE-RhlR interaction. A loop rearrangement that repositions the E280 residue in PqsE(E182W) is responsible for the loss of interaction. We verify the implications garnered from the PqsE(E182W) structure using mutagenic, biochemical, and additional structural analyses. We present the next generation of molecules targeting the PqsE active site, including a structure of the tightest binding of these compounds, BB584, in complex with PqsE. The findings presented here provide insight for drug discovery againstP. aeruginosawith PqsE as the target.Author SummaryThe human pathogenPseudomonas aeruginosais resistant to many currently used antibiotics, making it a burden of urgent clinical importance.P. aeruginosapathogenicity is controlled by the bacterial cell-to-cell communication process called quorum sensing (QS). The function of one protein that controlsP. aeruginosaQS-directed virulence, RhlR, requires a protein-protein interaction with an enzyme called PqsE. When PqsE is blocked from interacting with RhlR,P. aeruginosais avirulent and incapable of infecting an animal host. Here, we validate the PqsE-RhlR interaction as a target for antibiotic development, and we present a mechanism for how such antibiotics could disrupt the PqsE-RhlR interaction. Discovery of new antibiotics would fulfill an unmet healthcare need by providing treatments to combatP. aeruginosainfections.