Sialylation of Outer Membrane Porin Protein D: A Mechanistic Basis of Antibiotic Uptake in Pseudomonas aeruginosa

• Published in: Molecular & cellular proteomics Vol. 13; no. 6; pp. 1412 - 1428
• Main Authors: Khatua, Biswajit, Vleet, Jeremy Van, Choudhury, Biswa Pronab, Chaudhry, Rama, Mandal, Chitra
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2014; The American Society for Biochemistry and Molecular Biology
• Subjects: Anti-Bacterial Agents - administration & dosage; beta-Lactams - administration & dosage; beta-Lactams - chemistry; Humans; Polysaccharides - metabolism; Porins - chemistry; Porins - genetics; Porins - metabolism; Pseudomonas aeruginosa - drug effects; Pseudomonas aeruginosa - metabolism; Pseudomonas aeruginosa - pathogenicity; Pseudomonas Infections - drug therapy; Pseudomonas Infections - metabolism; Pseudomonas Infections - microbiology; Sialic Acids - metabolism
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M113.030999

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.