Systematic Mutational Analysis of the Putative Hydrolase PqsE: Toward a Deeper Molecular Understanding of Virulence Acquisition in Pseudomonas aeruginosa

Benjamin Folch,Nicolas Doucet,Eric Déziel

doi:10.1371/journal.pone.0073727

Benjamin Folch, Nicolas Doucet + Show 1 more

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https://doi.org/10.1371/journal.pone.0073727

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Abstract

Pseudomonas aeruginosa is an important opportunistic human pathogen that can establish bacterial communication by synchronizing the behavior of individual cells in a molecular phenomenon known as “quorum sensing”. Through an elusive mechanism involving gene products of the pqs operon, the PqsE enzyme is absolutely required for the synthesis of extracellular phenazines, including the toxic blue pigment pyocyanin, effectively allowing cells to achieve full-fledged virulence. Despite several functional and structural attempts at deciphering the role of this relevant enzymatic drug target, no molecular function has yet been ascribed to PqsE. In the present study, we report a series of alanine scanning experiments aimed at altering the biological function of PqsE, allowing us to uncover key amino acid positions involved in the molecular function of this enzyme. We use sequence analysis and structural overlays with members of homologous folds to pinpoint critical positions located in the vicinity of the ligand binding cleft and surrounding environment, revealing the importance of a unique C-terminal α-helical motif in the molecular function of PqsE. Our results suggest that the active site of the enzyme involves residues that extend further into the hydrophobic core of the protein, advocating for a lid-like movement of the two terminal helices. This information should help design virtual libraries of PqsE inhibitors, providing means to counter P. aeruginosa virulence acquisition and helping to reduce nosocomial infections.

Highlights

Pseudomonas aeruginosa is a prevalent opportunistic pathogen that can trigger serious infections in a wide variety of hosts, including plants, insects, and animals [1]
P. aeruginosa infections are linked to the synthesis of various virulence factors such as proteases, rhamnolipids, hydrogen cyanide, exotoxins, and phenazines, which cause a number of inflammatory and oxidative stresses leading to dysfunction of the respiratory tract [7]
The PqsE protein from P. aeruginosa is encoded by the pqsABCDE operon, which is required for the synthesis of the small diffusible molecules alkyl-4 (1H)-quinolones (AQs), including the signals HHQ (2-heptyl-4-hydroxyquinolone) and PQS (2-heptyl-3-hydroxy-4-quinolone)

Summary

Introduction

Pseudomonas aeruginosa is a prevalent opportunistic pathogen that can trigger serious infections in a wide variety of hosts, including plants, insects, and animals [1]. P. aeruginosa is a major nosocomial pathogen responsible for many hospital and clinical outbreaks worldwide [2] It is responsible for persistent drug refractile lung infections [3], sepsis in burn patients [4], as well as morbidity and mortality among individuals suffering from cystic fibrosis [5,6]. This pathogen is difficult to eradicate due to its high level of antibiotic resistance, which involves a variety of molecular mechanisms including multidrug efflux pumps, outermembrane porins, and inactivating enzymes [7]. To efficiently regulate the expression of most of its virulence factors, P. aeruginosa employs a quorum sensing regulatory network comprising at least three cell-to-cell signaling systems [8,9]: two acyl-homoserine lactone (AHL)-based LuxRI quorum-sensing systems and the MvfR (PqsR) system functioning through 2-alkyl-4 (1H)-quinolones (AQs) (such as the Pseudomonas Quinolone Signal-PQS)

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