title: Sialylation of outer membrane porin protein D: a mechanistic basis of antibiotic uptake in Pseudomonas aeruginosa.
abstract: 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.
authors.affiliation: From the ‡Cancer 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;
authors.forename: Biswajit
authors.initials: B
authors.lastname: Khatua
authors.affiliation: §Glycobiology Core Resources, Regents of the University of California, USCD, 9500 Gilman Drive (MC-0687), La Jolla, California, 92093-0687;
authors.forename: Jeremy
authors.initials: J
authors.lastname: Van Vleet
authors.affiliation: §Glycobiology Core Resources, Regents of the University of California, USCD, 9500 Gilman Drive (MC-0687), La Jolla, California, 92093-0687;
authors.forename: Biswa Pronab
authors.initials: BP
authors.lastname: Choudhury
authors.affiliation: ¶Microbiology Department, All India Institute of Medical Sciences, Ansari Nagar East, Gautam Nagar, New Delhi, Delhi 110029.
authors.forename: Rama
authors.initials: R
authors.lastname: Chaudhry
authors.affiliation: From the ‡Cancer 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.
authors.forename: Chitra
authors.initials: C
authors.lastname: Mandal
mesh_terms: D000900:Anti-Bacterial Agents / Q000008:administration & dosage; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism; D018272:Porins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011552:Pseudomonas Infections / Q000188:drug therapy* / Q000378:metabolism / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000378:metabolism* / Q000472:pathogenicity; D012794:Sialic Acids / Q000378:metabolism*; D047090:beta-Lactams / Q000008:administration & dosage / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D011134:Polysaccharides; D018272:Porins; D012794:Sialic Acids; D047090:beta-Lactams; C077157:OprD protein, Pseudomonas aeruginosa