Molecular Insights into Function and Competitive Inhibition of Pseudomonas aeruginosa Multiple Virulence Factor Regulator

Abstract

New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by multidrug-resistant (MDR) bacteria. Multiple virulence factor regulator (MvfR or PqsR), a Pseudomonas aeruginosa quorum sensing transcription factor, regulates functions important in both acute and persistent infections. Recently identified non-ligand-based benzamine-benzimidazole (BB) inhibitors of MvfR suppress both acute and persistent P. aeruginosa infections in mice without perturbing bacterial growth. Here, we elucidate the crystal structure of the MvfR ligand binding domain (LBD) in complex with one potent BB inhibitor, M64. Structural analysis indicated that M64 binds, like native ligands, to the MvfR hydrophobic cavity. A hydrogen bond and pi interaction were found to be important for MvfR-M64 affinity. Surface plasmon resonance analysis demonstrated that M64 is a competitive inhibitor of MvfR. Moreover, a protein engineering approach revealed that Gln194 and Tyr258 are critical for the interaction between MvfR and M64. Random mutagenesis of the full-length MvfR protein identified a single-amino-acid substitution, I68F, at a DNA binding linker domain that confers M64 insensitivity. In the presence of M64, I68F but not the wild-type (WT) MvfR protein retained DNA binding ability. Our findings strongly suggest that M64 promotes conformational change at the DNA binding domain of MvfR and that the I68F mutation may compensate for this change, indicating allosteric inhibition. This work provides critical new insights into the molecular mechanism of MvfR function and inhibition that could aid in the optimization of anti-MvfR compounds and improve our understanding of MvfR regulation.

The Study of Mutation in LasR and PqsR Genes in Extremely Drug Resistant and Multidrug Resistant Strains of Pseudomonas aeruginosa from Burn Wound Infection

“…The results of this investigation indicated that although Q194E retained at least 88% functionality, this mutant was virtually inactive and almost failed to respond to the Pseudomonas quinolone signal in the pqsA mutant. On the other hand, a hydrophobic amino acid at position 258 is required for PqsR functionality since Y258A mutation renders PqsR inactive (35). No mutations at positions 194 and 258 were seen in our study, but the only missense mutation in the present study (A314V) increased protein stability along with having a neutral impact on protein function.…”

Antivirulence properties and related mechanisms of spice essential oils: A comprehensive review

“…At present, the QS system (Figure 3) is more deeply understood in P. aeruginosa , which harbors four types of QS systems, including two LuxI/LuxR‐type (LasI/LasR and RhlI/RhlR) systems, a pseudomonas quinolone signal (PQS) system, and an integrating quorum sensing signal (IQS) system, all of which influence almost 10% of gene expression in P. aeruginosa , constituting a hierarchical regulatory network (Shrikant & Chandrajit, 2018). These four types of systems synthesize N‐3‐oxododecanyol‐L‐homoserine lactone (3OC12HSL), N‐butanoyl‐L‐homoserine lactone (C4‐HSL), 2‐heptyl‐3‐hydroxy‐4‐quinolone (PQS), and IQS as respective signal molecules to form complexes with corresponding receptors, and then activate genes encoding virulence factors such as elastase, proteases, rhamnolipids, hemolysins, toxins, pyocyanin, and alginate, which usually damage tissues and organs and interfere with the defense mechanisms of the human immune system, triggering different types of infection (Guo et al., 2014; Kitao et al., 2018). Moreover, swarming motility and biofilm formation are also under the control of QS system in P. aeruginosa (Rutherford & Bassler, 2012).…”

Cladodionen Is a Potential Quorum Sensing Inhibitor Against Pseudomonas aeruginosa

“…PqsR (MvfR) is a key component of alkyl-quinolone-dependent QS in P. aeruginosa [13]. The LBD of PqsR consists of two subdomains connected by an antiparallel β-sheet hinge region, with a large ligand binding pocket in which a native agonist 2-nonyl-4-quinolone (NHQ) is stabilized entirely by hydrophobic interactions [13,14]. Residues Leu189, Leu207, Leu208, Ala168, Ile149, Ile236, Tyr258, Ile186 and Val170 take part in the above hydrophobic interactions between PqsR and NHQ.…”