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Structural basis for the interaction of lipopolysaccharide with outer membrane protein H (OprH) from Pseudomonas aeruginosa.

ABSTRACT: Pseudomonas aeruginosa is a major nosocomial pathogen that infects cystic fibrosis and immunocompromised patients. The impermeability of the P. aeruginosa outer membrane contributes substantially to the notorious antibiotic resistance of this human pathogen. This impermeability is partially imparted by the outer membrane protein H (OprH). Here we have solved the structure of OprH in a lipid environment by solution NMR. The structure reveals an eight-stranded ?-barrel protein with four extracellular loops of unequal size. Fast time-scale dynamics measurements show that the extracellular loops are disordered and unstructured. It was previously suggested that the function of OprH is to provide increased stability to the outer membranes of P. aeruginosa by directly interacting with lipopolysaccharide (LPS) molecules. Using in vivo and in vitro biochemical assays, we show that OprH indeed interacts with LPS in P. aeruginosa outer membranes. Based upon NMR chemical shift perturbations observed upon the addition of LPS to OprH in lipid micelles, we conclude that the interaction is predominantly electrostatic and localized to charged regions near both rims of the barrel, but also through two conspicuous tyrosines in the middle of the bilayer. These results provide the first molecular structure of OprH and offer evidence for multiple interactions between OprH and LPS that likely contribute to the antibiotic resistance of P. aeruginosa.

SUBMITTER: Edrington TC

PROVIDER: S-EPMC3234746 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Structural basis for the interaction of lipopolysaccharide with outer membrane protein H (OprH) from Pseudomonas aeruginosa.

Edrington Thomas C TC Kintz Erica E Goldberg Joanna B JB Tamm Lukas K LK

The Journal of biological chemistry 20110824 45

Pseudomonas aeruginosa is a major nosocomial pathogen that infects cystic fibrosis and immunocompromised patients. The impermeability of the P. aeruginosa outer membrane contributes substantially to the notorious antibiotic resistance of this human pathogen. This impermeability is partially imparted by the outer membrane protein H (OprH). Here we have solved the structure of OprH in a lipid environment by solution NMR. The structure reveals an eight-stranded β-barrel protein with four extracellu ...[more]

PMID: 21865172

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Project description:Pseudomonas aeruginosa (PA) is an environmentally ubiquitous, extracellular, opportunistic pathogen, associated with severe infections of immune-compromised host. We demonstrated earlier the presence of both α2,3- and α2,6-linked sialic acids (Sias) on PA (PA(+Sias)) and normal human serum is their source of Sias. PA(+Sias) showed decreased complement deposition and exhibited enhanced association with immune-cells through sialic acid binding immunoglobulin like lectins (Siglecs). Such Sias-siglec-9 interaction between PA(+Sias) and neutrophils helped to subvert host immunity. Additionally, PA(+Sias) showed more resistant to β-lactam antibiotics as reflected in their minimum inhibitory concentration required to inhibit the growth of 50% than PA(-Sias). Accordingly, we have affinity purified sialoglycoproteins of PA(+Sias). They were electrophoresed and identified by matrix-assisted laser desorption-ionization time-of-flight/time-of-flight mass spectrometry analysis. Sequence study indicated the presence of a few α2,6-linked, α2,3-linked, and both α2,3- and α2,6-linked sialylated proteins in PA. The outer membrane porin protein D (OprD), a specialized channel-forming protein, responsible for uptake of β-lactam antibiotics, is one such identified sialoglycoprotein. Accordingly, sialylated (OprD(+Sias)) and non-sialylated (OprD(-Sias)) porin proteins were separately purified by using anion exchange chromatography. Sialylation of purified OprD(+Sias) was confirmed by several analytical and biochemical procedures. Profiling of glycan structures revealed three sialylated N-glycans and two sialylated O-glycans in OprD(+Sias). In contrast, OprD(-Sias) exhibit only one sialylated N-glycans. OprD(-Sias) interacts with β-lactam antibiotics more than OprD(+Sias) as demonstrated by surface plasmon resonance study. Lyposome-swelling assay further exhibited that antibiotics have more capability to penetrate through OprD(-Sias) purified from four clinical isolates of PA. Taken together, it may be envisaged that sialic acids on OprD protein play important role toward the uptake of commonly used antibiotics in PA(+Sias). This might be one of the new mechanisms of PA for β-lactam antibiotic uptake.

Dataset Information

Structural basis for the interaction of lipopolysaccharide with outer membrane protein H (OprH) from Pseudomonas aeruginosa.

Publications

Structural basis for the interaction of lipopolysaccharide with outer membrane protein H (OprH) from Pseudomonas aeruginosa.

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