title: Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species.
abstract: Many human-dwelling bacteria acquire sialic acid for growth or surface display. We identified previously a sialic acid utilization operon in Tannerella forsythia that includes a novel outer membrane sialic acid-transport system (NanOU), where NanO (neuraminate outer membrane permease) is a putative TonB-dependent receptor and NanU (extracellular neuraminate uptake protein) is a predicted SusD family protein. Using heterologous complementation of nanOU genes into an Escherichia coli strain devoid of outer membrane sialic acid permeases, we show that the nanOU system from the gut bacterium Bacteroides fragilis is functional and demonstrate its dependence on TonB for function. We also show that nanU is required for maximal function of the transport system and that it is expressed in a sialic acid-responsive manner. We also show its cellular localization to the outer membrane using fractionation and immunofluorescence experiments. Ligand-binding studies revealed high-affinity binding of sialic acid to NanU (Kd ~400 nM) from two Bacteroidetes species as well as binding of a range of sialic acid analogues. Determination of the crystal structure of NanU revealed a monomeric SusD-like structure containing a novel motif characterized by an extended kinked helix that might determine sugar-binding specificity. The results of the present study characterize the first bacterial extracellular sialic acid-binding protein and define a sialic acid-specific PUL (polysaccharide utilization locus).
authors.affiliation: *School of Clinical Dentistry, University of Sheffield, Sheffield S10 2TA, U.K.
authors.forename: Chatchawal
authors.initials: C
authors.lastname: Phansopa
authors.affiliation: *School of Clinical Dentistry, University of Sheffield, Sheffield S10 2TA, U.K.
authors.forename: Sumita
authors.initials: S
authors.lastname: Roy
authors.affiliation: †Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
authors.forename: John B
authors.initials: JB
authors.lastname: Rafferty
authors.affiliation: *School of Clinical Dentistry, University of Sheffield, Sheffield S10 2TA, U.K.
authors.forename: C W Ian
authors.initials: CW
authors.lastname: Douglas
authors.affiliation: ‡Department of Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD, U.K.
authors.forename: Jagroop
authors.initials: J
authors.lastname: Pandhal
authors.affiliation: ‡Department of Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD, U.K.
authors.forename: Phillip C
authors.initials: PC
authors.lastname: Wright
authors.affiliation: †Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
authors.forename: David J
authors.initials: DJ
authors.lastname: Kelly
authors.affiliation: *School of Clinical Dentistry, University of Sheffield, Sheffield S10 2TA, U.K.
authors.forename: Graham P
authors.initials: GP
authors.lastname: Stafford
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001441:Bacteroides fragilis / Q000235:genetics / Q000378:metabolism; D041963:Bacteroidetes / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D007422:Intestines / Q000382:microbiology; D008565:Membrane Proteins / Q000378:metabolism*; D009055:Mouth / Q000382:microbiology; D009154:Mutation; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008565:Membrane Proteins; C057616:tonB protein, Bacteria; D019158:N-Acetylneuraminic Acid