title: Identification of major outer surface proteins of Streptococcus agalactiae.
abstract: To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates.
authors.affiliation: Microscience Ltd, Wokingham, Berks, United Kingdom. m.hughes@microscience.com
authors.forename: Martin J G
authors.initials: MJ
authors.lastname: Hughes
authors.forename: Joanne C
authors.initials: JC
authors.lastname: Moore
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Lane
authors.forename: Rebecca
authors.initials: R
authors.lastname: Wilson
authors.forename: Philippa K
authors.initials: PK
authors.lastname: Pribul
authors.forename: Zabin N
authors.initials: ZN
authors.lastname: Younes
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Dobson
authors.forename: Paul
authors.initials: P
authors.lastname: Everest
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Reason
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Redfern
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Greer
authors.forename: Thanai
authors.initials: T
authors.lastname: Paxton
authors.forename: Maria
authors.initials: M
authors.lastname: Panico
authors.forename: Howard R
authors.initials: HR
authors.lastname: Morris
authors.forename: Robert G
authors.initials: RG
authors.lastname: Feldman
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Santangelo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D000907:Antibodies, Bacterial / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional; D007116:Immunization, Passive; D051379:Mice; D008969:Molecular Sequence Data; D009954:Ornithine Carbamoyltransferase / Q000276:immunology; D010735:Phosphoglycerate Kinase / Q000276:immunology; D020543:Proteome; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013290:Streptococcal Infections / Q000209:etiology / Q000517:prevention & control; D013292:Streptococcus agalactiae / Q000737:chemistry* / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D020543:Proteome; D009954:Ornithine Carbamoyltransferase; D010735:Phosphoglycerate Kinase