Molecular Basis for the Interaction of Lipopolysaccharide with Outer Membrane Protein OprH from Pseudomonas Aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic 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 OprH. The function of OprH is to provide increased stability to the outer membranes of P. aeruginosa by directly interacting with LPS molecules. The NMR solution structure of OprH reveals an eight-stranded β-barrel with four extracellular loops of unequal size (Edrington et al, J. Biol. Chem. 286:39211[2011]). Based on NMR chemical shift perturbations observed upon the addition of LPS to OprH in lipid micelles, we concluded that the interaction is predominantly electrostatic and localized to charged regions near upper rims of the barrel and the two shortest loops. Using an enzyme-linked immunosorbent assay (ELISA) we discovered that the intact secondary structure is necessary for OprH to interact with LPS, and that the interaction occurs mainly through the charged side chains of K70, R72, K103 and R113. Despite detailed analysis of NMR spectra upon the addition of LPS to OprH, we found no evidence for an interaction of the hydrophobic side-chains of I, L and V with lipid A of LPS. The results of this study provide a more definitive molecular model for interactions between OprH and LPS and offer new insight into protein-lipid interactions that likely contribute to the antibiotic resistance during P. aeruginosa infections.