Taylor, I. R. | Search bioRxiv

bioRxiv2024

PqsE acts as an adaptor protein for the quorum-sensing transcription factor RhlR in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a human pathogen that poses a significant health threat. Pathogenic behaviors of P. aeruginosa are under control of the bacterial cell-cell communication system known as quorum sensing (QS). One of the QS master regulators, RhlR, is a receptor/transcription factor that not only relies on binding of its canonical ligand, C4-homoserine lactone (HSL), but additionally requires a protein-protein interaction with the enzyme, PqsE. We constructed heterologous reporter strains in Escherichia coli that allow measurements of the reliance of RhlR on C4-HSL and/or PqsE-binding for the ability to activate transcription of three RhlR-regulated genes: rhlA (PqsE-independent), phzM (PqsE-dependent), and azeB (PqsE-suppressed). Analogous assays measuring activation of the three genes in P. aeruginosa were performed and the patterns observed correlated tightly with the heterologous reporter assays. These results indicate that binding of PqsE to RhlR is able to fine-tune RhlR transcription factor activity in a promoter-specific manner. ImportancePseudomonas aeruginosa is an opportunistic human pathogen that can cause fatal infections. There exists an urgent need for new, effective antimicrobial agents to combat P. aeruginosa. The PqsE-RhlR protein-protein interaction is essential for P. aeruginosa to be able to make toxins, form biofilms, and infect host organisms. In this study, we use both non-native models in E. coli and measurements of gene expression/toxin production in P. aeruginosa to show that the PqsE-RhlR interaction enables fine-tuned gene expression and a heightened ability of P. aeruginosa to adapt to external conditions. These findings will be highly valuable as continued efforts are made to design inhibitors of the PqsE-RhlR interaction and test them as potential antimicrobial agents against P. aeruginosa infections.

microbiology↗

bioRxiv2022

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

The opportunistic pathogen Pseudomonas aeruginosa causes antibiotic resistant, nosocomial infections in immuno-compromised individuals, and is a high priority for antimicrobial development. Key to pathogenicity in P. aeruginosa are 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, attenuates P. aeruginosa infection 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 against P. aeruginosa with PqsE as the target. Author SummaryThe human pathogen Pseudomonas aeruginosa is resistant to many currently used antibiotics, making it a burden of urgent clinical importance. P. aeruginosa pathogenicity is controlled by the bacterial cell-to-cell communication process called quorum sensing (QS). The function of one protein that controls P. aeruginosa QS-directed virulence, RhlR, requires a protein-protein interaction with an enzyme called PqsE. When PqsE is blocked from interacting with RhlR, P. aeruginosa is 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 combat P. aeruginosa infections.

microbiology↗