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

作者：Khatua Biswajit; Van Vleet Jeremy; Choudhury Biswa Pronab; Chaudhry Rama; Mandal Chitra^*

来源：Molecular & Cellular Proteomics, 2014, 13(6): 1412-1428.

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 alpha 2,3-and alpha 2,6-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 beta-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 alpha 2,6-linked, alpha 2,3-linked, and both alpha 2,3-and alpha 2,6-linked sialylated proteins in PA. The outer membrane porin protein D (OprD), a specialized channel-forming protein, responsible for uptake of beta-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 beta-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 beta-lactam antibiotic uptake.

• 出版日期2014-6
• 单位河北医科大学