The PqsE-RhlR Interaction Regulates RhlR DNA Binding to Control Virulence Factor Production in
            <i>Pseudomonas aeruginosa</i>

Simanek, Kayla A; Taylor, Isabelle R.; Richael, Erica K; Lasek‐Nesselquist, Erica; Bassler, Bonnie L.; Paczkowski, Jon E.

doi:10.1128/spectrum.02108-21

Cited by 45 publications

(118 citation statements)

References 47 publications

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“…Deletion of the gene encoding the enzyme, PqsE, that acts in a different P. aeruginosa QS pathway, results in the identical loss of pathogenic phenotypes as occur following deletion of rhlR (8). Consistent with this result, we recently showed that PqsE and RhlR make a protein-protein interaction that enhances the affinity of RhlR for target DNA sequences (9, 10). The PqsE-RhlR interaction is, moreover, essential for RhlR-controlled QS phenotypes.…”

Section: Observationsupporting

confidence: 71%

The Protein Interaction Networks of Catalytically-Active and Catalytically-Inactive PqsE in Pseudomonas aeruginosa

Taylor

Murray-Nerger

Cristea

et al. 2022

Preprint

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Pseudomonas aeruginosa is a human pathogen that relies on quorum sensing to establish infections. The PqsE quorum-sensing protein is required for P. aeruginosa virulence factor production and infection. PqsE has a reported enzymatic function in the biosynthesis of the quorum-sensing autoinducer called PQS. However, this activity is redundant because, in the absence of PqsE, this role is fulfilled by alternative thioesterases. Rather, PqsE drives P. aeruginosa pathogenic traits via a protein-protein interaction with the quorum-sensing receptor/transcription factor, RhlR, an interaction that enhances affinity of RhlR for target DNA sequences. PqsE catalytic activity is dispensable for interaction with RhlR. Thus, PqsE virulence function can be decoupled from its catalytic function. Here, we present an immunoprecipitation-mass spectrometry method employing eGFP-PqsE fusions to define the protein interactomes of wildtype PqsE and the catalytically inactive PqsE(D73A) variant in P. aeruginosa and their dependence on RhlR. Several proteins were identified to have specific interactions with wildtype PqsE, while not forming associations with PqsE(D73A). In the ΔrhlR strain, an increased number of specific PqsE interactors were identified, including the partner autoinducer synthase to RhlR, called RhlI. Collectively, these results suggest that specific protein-protein interactions depend on PqsE catalytic activity and that RhlR may prevent proteins from interacting with PqsE, possibly due to competition between RhlR and other proteins for PqsE binding. Our results provide a foundation for the identification of the in vivo PqsE catalytic function and, potentially, new proteins involved in P. aeruginosa quorum sensing.

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Section: Observationsupporting

confidence: 71%

The Protein Interaction Networks of Catalytically-Active and Catalytically-Inactive PqsE in Pseudomonas aeruginosa

Taylor

Murray-Nerger

Cristea

et al. 2022

Preprint

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“…Our finding that the PqsE-RhlR interaction was disrupted following introduction of the E182W substitution in the PqsE active site was surprising given that this residue is distal to the RhlR interaction site (21). The crystal structure of PqsE(E182W) revealed that a loop rearrangement inserts the E280 residue into the active site to coordinate both of the iron atoms at this site.…”

Section: Discussionmentioning

confidence: 91%

“…6xHis-PqsE proteins were purified for biochemical assays (tagged) and crystallography (tag removed) as described previously (20). Enzyme activities of purified 6xHis-PqsE variants were measured as previously reported with 4-methylumbelliferyl butyrate (MU-butyrate) as the substrate (21). Fluorescence polarization assays with the BB562 active site probe were performed as described (20).…”

Section: Methodsmentioning

confidence: 99%

“…Curiously, RhlR function additionally depends on the metallo-𝛽hydrolase enzyme PqsE, encoded as the final gene in the pqsABCDE biosynthetic operon (17)(18)(19). With respect to RhlR, PqsE is required to form a protein-protein interaction that enhances the affinity of RhlR for target promoters (20,21). Moreover, the RhlR-PqsE interaction is essential for P. aeruginosa virulence phenotypes, and therefore, of interest as a focus for antibiotic development.…”

Section: Introductionmentioning

confidence: 99%

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

Taylor

Jeffrey

Moustafa

et al. 2022

Preprint

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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.

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“…In addition to the LasI/LasR and RhlI/RhlR QS systems, the 2-heptyl-3-hydroxy-4(1H)-quinolone ( Pseudomonas quinolone signal, PQS) circuit is the third system regulated by PqsR, which relies on PQS. Recently, many studies reported the interaction between Rhl and Pqs systems [ 9 , 10 , 11 ]. RhlR negatively regulates the expression of pqsABCDE operon independently of PQS production.…”

Section: Introductionmentioning

confidence: 99%

Recent Advance in Small Molecules Targeting RhlR of Pseudomonas aeruginosa

et al. 2022

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Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic gram-negative pathogen that can cause various infections, particularly in patients with compromised host defenses. P. aeruginosa forms biofilms and produces virulence factors through quorum sensing (QS) network, resulting in resistance to antibiotics. RhlI/RhlR, one of key QS systems in P. aeruginosa, is considered an attractive target for inhibiting biofilm formation and attenuating virulence factors. Several recent studies examined small molecules targeting the RhlI/RhlR system and their in vitro and in vivo biological activities. In this review, RhlR-targeted modulators, including agonists and antagonists, are discussed with particular focus on structure-activity relationship studies and outlook for next-generation anti-biofilm agents.

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The PqsE-RhlR Interaction Regulates RhlR DNA Binding to Control Virulence Factor Production in Pseudomonas aeruginosa

Abstract: Bacteria use a cell-cell communication process called quorum sensing (QS) to orchestrate collective behaviors. QS relies on the group-wide detection of molecules called autoinducers (AI).

Cited by 45 publications

References 47 publications

The Protein Interaction Networks of Catalytically-Active and Catalytically-Inactive PqsE in Pseudomonas aeruginosa

The Protein Interaction Networks of Catalytically-Active and Catalytically-Inactive PqsE in Pseudomonas aeruginosa

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

Recent Advance in Small Molecules Targeting RhlR of Pseudomonas aeruginosa

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