Identification of major outer surface proteins of Streptococcus agalactiae.
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.
Hughes
Martin J G
MJ
Microscience Ltd, Wokingham, Berks, United Kingdom. m.hughes@microscience.com
Moore
Joanne C
JC
Lane
Jonathan D
JD
Wilson
Rebecca
R
Pribul
Philippa K
PK
Younes
Zabin N
ZN
Dobson
Richard J
RJ
Everest
Paul
P
Reason
Andrew J
AJ
Redfern
Joanne M
JM
Greer
Fiona M
FM
Paxton
Thanai
T
Panico
Maria
M
Morris
Howard R
HR
Feldman
Robert G
RG
Santangelo
Joseph D
JD
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
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D020543:Proteome; D009954:Ornithine Carbamoyltransferase; D010735:Phosphoglycerate Kinase