Biology subjects

Taylor, I. R.

Publications and source records attributed to Taylor, I. R..

4 recordsLinked to original sources

bioRxiv2025

New inhibitors of the Pseudomonas aeruginosa enzyme, PqsE, and methods assessing their potential to induce a conformational change via active site binding

Pseudomonas aeruginosa is an opportunistic pathogen known for its ability to produce virulence factors and form biofilms. These, among many other traits, enable P. aeruginosa to cause infections, evade host immune responses, and resist treatment with antimicrobial agents. Both the ability to form biofilms and produce virulence factors are regulated via the bacterial cell-cell communication process called quorum sensing. A key molecular event that enables quorum sensing in P. aeruginosa is the physical interaction between an enzyme, PqsE and a master quorum-sensing receptor/transcription factor RhlR, which regulates the expression of a wide variety of virulence-associated genes. Previous work identified active site mutations in PqsE that induce a conformational change weakening the interaction with RhlR. These active site mutations weakened the PqsE-RhlR interaction to the extent that several virulence genes were not activated, and the mutant strains of P. aeruginosa failed to colonize the lungs of a mouse. In this study, we designed a series of molecules to probe binding in the active site of PqsE as a strategy for inhibiting the PqsE-RhlR interaction and thus, the activation of RhlR-controlled genes in P. aeruginosa. HJ1 and HJ5 are new molecules that both bind in the active site of PqsE, and while HJ5 appears to bind in an alternate mode compared to HJ1, it does not induce a conformational change to weaken the PqsE-RhlR interaction. Here, we introduce multiple experimental approaches to assess the way in which these new molecules engage in the PqsE active site. HJ5 can serve as a promising starting point for the development of molecules that target the PqsE active site and allosterically inhibit the interaction with RhlR, thus decreasing virulence in P. aeruginosa.

biochemistry↗

bioRxiv2025

Screening of FDA-approved small molecules to discover inhibitors of the Pseudomonas aeruginosa quorum-sensing enzyme, PqsE

Pseudomonas aeruginosa is a notorious pathogen that is a leading cause of hospital-acquired infections. Due to its heightened resistance to a broad range of antibiotics, there is great need for new antimicrobial agents that are effective in treating P. aeruginosa infections. One attractive option for exploring new antibiotic targets is the quorum sensing (QS) pathway, as it governs many of the pathogenic behaviors that allow P. aeruginosa to stage and maintain infections. Within the QS pathway, there is a key protein-protein interaction between an enzyme, PqsE, and one of the master QS regulators, RhlR. Although its catalytic function is dispensable for its interaction with RhlR, previous mutagenic work characterizing the active site of PqsE identified active site mutations that induce a conformational change in PqsE, preventing it from forming a complex with RhlR. These active site mutations, when introduced stably into the genome of P. aeruginosa, also lead to a significant decrease in production of a key toxin, pyocyanin, and prevent colonization in the lungs of a murine host. These findings encourage drug discovery efforts to identify small molecules that bind in the active site of PqsE and induce a conformational change similar to the ones induced by particular amino acid substitutions in the active site. Here, we performed a fluorescence polarization screen to identify molecules that bind in the active site of PqsE. We screened a library of FDA-approved drugs, with the intention of potentially repurposing a known molecule for the treatment of P. aeruginosa infections. Three molecules were identified, two of which showed inhibitory activity that was consistent with a competitive mode of inhibition. One hit molecule, Apomorphine, had a distinctly different inhibitory profile, and is potentially binding outside of the active site to allosterically inhibit enzyme activity of PqsE. All three hit molecules were tested in an in vivo enzyme activity assay, and one of the competitive inhibitors, Vorinostat, was found to inhibit intracellular PqsE. Vorinostat is now being explored as a candidate for synthetic derivatization to inhibit the PqsE-RhlR protein-protein interaction via binding in the PqsE active site.

biochemistry↗

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↗