PMID- 24643970 OWN - NLM STAT- MEDLINE DCOM- 20150219 LR - 20211021 IS - 1535-9484 (Electronic) IS - 1535-9476 (Print) IS - 1535-9476 (Linking) VI - 13 IP - 6 DP - 2014 Jun TI - Sialylation of outer membrane porin protein D: a mechanistic basis of antibiotic uptake in Pseudomonas aeruginosa. PG - 1412-28 LID - 10.1074/mcp.M113.030999 [doi] AB - 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 alpha2,3- and alpha2,6-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 alpha2,6-linked, alpha2,3-linked, and both alpha2,3- and alpha2,6-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. CI - (c) 2014 by The American Society for Biochemistry and Molecular Biology, Inc. FAU - Khatua, Biswajit AU - Khatua B AD - From the double daggerCancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700 032, India; FAU - Van Vleet, Jeremy AU - Van Vleet J AD - section signGlycobiology Core Resources, Regents of the University of California, USCD, 9500 Gilman Drive (MC-0687), La Jolla, California, 92093-0687; FAU - Choudhury, Biswa Pronab AU - Choudhury BP AD - section signGlycobiology Core Resources, Regents of the University of California, USCD, 9500 Gilman Drive (MC-0687), La Jolla, California, 92093-0687; FAU - Chaudhry, Rama AU - Chaudhry R AD - paragraph signMicrobiology Department, All India Institute of Medical Sciences, Ansari Nagar East, Gautam Nagar, New Delhi, Delhi 110029. FAU - Mandal, Chitra AU - Mandal C AD - From the double daggerCancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700 032, India; chitra_mandal@yahoo.com cmandal@iicb.res.in. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20140318 PL - United States TA - Mol Cell Proteomics JT - Molecular & cellular proteomics : MCP JID - 101125647 RN - 0 (Anti-Bacterial Agents) RN - 0 (Polysaccharides) RN - 0 (Porins) RN - 0 (Sialic Acids) RN - 0 (beta-Lactams) RN - 148412-32-2 (OprD protein, Pseudomonas aeruginosa) SB - IM MH - Anti-Bacterial Agents/administration & dosage MH - Humans MH - Polysaccharides/metabolism MH - Porins/chemistry/genetics/*metabolism MH - Pseudomonas Infections/*drug therapy/metabolism/microbiology MH - Pseudomonas aeruginosa/drug effects/*metabolism/pathogenicity MH - Sialic Acids/*metabolism MH - beta-Lactams/administration & dosage/chemistry PMC - PMC4047463 EDAT- 2014/03/20 06:00 MHDA- 2015/02/20 06:00 PMCR- 2015/06/01 CRDT- 2014/03/20 06:00 PHST- 2014/03/20 06:00 [entrez] PHST- 2014/03/20 06:00 [pubmed] PHST- 2015/02/20 06:00 [medline] PHST- 2015/06/01 00:00 [pmc-release] AID - S1535-9476(20)33074-7 [pii] AID - M113.030999 [pii] AID - 10.1074/mcp.M113.030999 [doi] PST - ppublish SO - Mol Cell Proteomics. 2014 Jun;13(6):1412-28. doi: 10.1074/mcp.M113.030999. Epub 2014 Mar 18.