title: Dissection of the function of the RmpM periplasmic protein from Neisseria meningitidis.
abstract: RmpM is a periplasmic protein from Neisseria meningitidis that comprises an N-terminal domain (residues 1-47) and a separate globular C-terminal domain (residues 65-219) responsible for binding to peptidoglycan. Here we show, through the use of size exclusion chromatography and pull-down assays, that a recombinant N-terminal fragment of RmpM binds to both the major outer membrane porins, PorA and PorB. Analysis by semi-native SDS-PAGE established that both recombinant full-length RmpM and an N-terminal fragment, but not the C-terminal peptidoglycan-binding domain, were sufficient to stabilize the PorA and PorB oligomeric complexes. Evidence from binding assays indicated that the meso-diaminopimelate moiety plays an important role in peptidoglycan recognition by RmpM. Site-directed mutagenesis showed that two highly conserved residues, Asp120 and Arg135, play an important role in peptidoglycan binding. The yield of outer membrane vesicles, which have been used extensively as a vaccine against N. meningitidis, was considerably higher in an N. meningitidis strain expressing a truncated N-terminal fragment of RmpM (ΔC-term rmpM) than in the WT strain. The native oligomeric state of the PorA/PorB complexes was maintained in this strain. We conclude that the dual functions of RmpM are independent, and that it is possible to use this knowledge to engineer a strain with higher yield of outer membrane vesicles, whilst preserving PorA and PorB, which are key protective antigens, in their native oligomeric state.
authors.affiliation: National Institute for Biological Standards and Control, South Mimms, Hertfordshire, EN6 3QGUK.|Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester, M13 9PTUK.
authors.forename: Sunil
authors.initials: S
authors.lastname: Maharjan
authors.affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester, M13 9PTUK.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Saleem
authors.affiliation: National Institute for Biological Standards and Control, South Mimms, Hertfordshire, EN6 3QGUK.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Feavers
authors.affiliation: National Institute for Biological Standards and Control, South Mimms, Hertfordshire, EN6 3QGUK.
authors.forename: Jun X
authors.initials: JX
authors.lastname: Wheeler
authors.affiliation: National Institute for Biological Standards and Control, South Mimms, Hertfordshire, EN6 3QGUK.
authors.forename: Rory
authors.initials: R
authors.lastname: Care
authors.affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester, M13 9PTUK.
authors.forename: Jeremy P
authors.initials: JP
authors.lastname: Derrick
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D003960:Diaminopimelic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016297:Mutagenesis, Site-Directed; D009345:Neisseria meningitidis / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism*; D018272:Porins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D010457:Peptidoglycan; D033903:Periplasmic Proteins; D018272:Porins; C499670:RmpM protein, Neisseria; C057699:porin protein, Neisseria; D003960:Diaminopimelic Acid