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Molecular conformation affects the interaction of the Pseudomonas quinolone signal with the bacterial outer membrane.

ABSTRACT: Gram-negative bacteria produce outer-membrane vesicles (OMVs) that package genetic elements, virulence factors, and cell-to-cell communication signaling compounds. Despite their importance in many disease-related processes, how these versatile structures are formed is incompletely understood. A self-produced secreted small molecule, the Pseudomonas quinolone signal (PQS), has been shown to initiate OMV formation in Pseudomonas aeruginosa by interacting with the outer membrane (OM) and inducing its curvature. Other bacterial species have also been shown to respond to PQS, supporting a common biophysical mechanism. Here, we conducted molecular dynamics simulations to elucidate the specific interactions between PQS and a model P. aeruginosa OM at the atomistic scale. We discovered two characteristic states of PQS interacting with the biologically relevant membrane, namely attachment to the membrane surface and insertion into the lipid A leaflet. The hydrogen bonds between PQS and the lipid A phosphates drove the PQS-membrane association. An analysis of PQS trajectory and molecular conformation revealed sequential events critical for spontaneous insertion, including probing, docking, folding, and insertion. Remarkably, PQS bent its hydrophobic side chain into a closed conformation to lower the energy barrier for penetration through the hydrophilic headgroup zone of the lipid A leaflet, which was confirmed by the potential of mean force (PMF) measurements. Attachment and insertion were simultaneously observed in the simulation with multiple PQS molecules. Our findings uncover a sequence of molecular interactions that drive PQS insertion into the bacterial OM and provide important insight into the biophysical mechanism of small molecule-induced OMV biogenesis.

SUBMITTER: Li A

PROVIDER: S-EPMC6349108 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Molecular conformation affects the interaction of the Pseudomonas quinolone signal with the bacterial outer membrane.

Li Ao A Schertzer Jeffrey W JW Yong Xin X

The Journal of biological chemistry 20181218 4

Gram-negative bacteria produce outer-membrane vesicles (OMVs) that package genetic elements, virulence factors, and cell-to-cell communication signaling compounds. Despite their importance in many disease-related processes, how these versatile structures are formed is incompletely understood. A self-produced secreted small molecule, the Pseudomonas quinolone signal (PQS), has been shown to initiate OMV formation in Pseudomonas aeruginosa by interacting with the outer membrane (OM) ...[more]

PMID: 30563840

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Project description:The Pseudomonas quinolone signal (PQS) is an important quorum-sensing molecule in Pseudomonas aeruginosa that also mediates its own packaging and transport by stimulating outer membrane vesicle (OMV) formation. Because OMVs have been implicated in many virulence-associated behaviors, it is critical that we understand how they are formed. Our group proposed the bilayer-couple model for OMV biogenesis, where PQS intercalates into the outer membrane, causing expansion of the outer leaflet and consequently inducing curvature. In accordance with the model, we hypothesized that PQS must be transported from the cytoplasm to the outer membrane before it can initiate OMV formation. We initially examined two laboratory strains of P. aeruginosa and found significant strain-dependent differences. PQS export correlated strongly with OMV production, even though equivalent amounts of total PQS were produced by both strains. Interestingly, we discovered that poor OMV producers sequestered the majority of PQS in the inner membrane, which appeared to be the result of early saturation of the export pathway. Further analysis showed that strain-specific PQS export and OMV biogenesis patterns were stable once established but could be significantly altered by changing the growth medium. Finally, we demonstrated that the associations described for laboratory strains also held for three clinical strains. These results suggest that factors controlling the export of PQS dictate OMV biogenesis. This work provides new insight into PQS-controlled virulence in P. aeruginosa and provides important tools to further study signal export and OMV biogenesis.IMPORTANCE Bacterial secretion has been recognized as an essential facet of microbial pathogenesis and human disease. Numerous virulence factors have been found to be transported within outer membrane vesicles (OMVs), and delivery using these biological nanoparticles often results in increased potency. OMV biogenesis is an important but poorly understood process that is ubiquitous among Gram-negative organisms. Our group seeks to understand the biochemical mechanisms behind the formation of OMVs and has developed a model of small-molecule-induced membrane curvature as an important driver of this process. With this work, we demonstrate that PQS, a known small-molecule OMV inducer, must be exported to promote OMV biogenesis in both lab-adapted and clinical strains of Pseudomonas aeruginosa In supporting and expanding the bilayer-couple model of OMV biogenesis, the current work lays the groundwork for studying environmental and genetic factors that modulate OMV production and, consequently, the packaging and delivery of many bacterial factors.

Project description:UnlabelledPseudomonas aeruginosa is a Gram-negative bacterium that is ubiquitous in the environment, and it is an opportunistic pathogen that can infect a variety of hosts, including humans. During the process of infection, P. aeruginosa coordinates the expression of numerous virulence factors through the production of multiple cell-to-cell signaling molecules. The production of these signaling molecules is linked through a regulatory network, with the signal N-(3-oxododecanoyl) homoserine lactone and its receptor LasR controlling the induction of a second acyl-homoserine lactone signal and the Pseudomonas quinolone signal (PQS). LasR-mediated control of PQS occurs partly by activating the transcription of pqsR, a gene that encodes the PQS receptor and is necessary for PQS production. We show that LasR interacts with a single binding site in the pqsR promoter region and that it does not influence the transcription of the divergently transcribed gene, nadA. Using DNA affinity chromatography, we identified additional proteins that interact with the pqsR-nadA intergenic region. These include the H-NS family members MvaT and MvaU, and CysB, a transcriptional regulator that controls sulfur uptake and cysteine biosynthesis. We show that CysB interacts with the pqsR promoter and that CysB represses pqsR transcription and PQS production. Additionally, we provide evidence that CysB can interfere with the activation of pqsR transcription by LasR. However, as seen with other CysB-regulated genes, pqsR expression was not differentially regulated in response to cysteine levels. These findings demonstrate a novel role for CysB in influencing cell-to-cell signal production by P. aeruginosa.ImportanceThe production of PQS and other 4-hydroxy-2-alkylquinolone (HAQs) compounds is a key component of the P. aeruginosa cell-to-cell signaling network, impacts multiple physiological functions, and is required for virulence. PqsR directly regulates the genes necessary for HAQ production, but little is known about the regulation of pqsR. We identified CysB as a novel regulator of pqsR and PQS production, but, unlike other CysB-controlled genes, it does not appear to regulate pqsR in response to cysteine. This implies that CysB functions as both a cysteine-responsive and cysteine-unresponsive regulator in P. aeruginosa.

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Molecular conformation affects the interaction of the Pseudomonas quinolone signal with the bacterial outer membrane.

Publications

Molecular conformation affects the interaction of the <i>Pseudomonas</i> quinolone signal with the bacterial outer membrane.

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