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.Streptococcus agalactiae (group B streptococcus [GBS]) is the causal agent of a broad range of human diseases. Of these, the most prominent are septicemia, pneumonia, and meningitis of neonates. The major component of the cell surface is a polysaccharide coat, which is serotype specific. Embedded throughout this coat is a complement of proteins. To date, only a few such proteins have been identified. Predominant of these are the tandem repeat proteins, including C␣ protein (40), R proteins (21), Rib protein (39), the C␤ protein (35), and X proteins (33).

Purification of Two Different Immunoglobulins (Igs) from Olive Flounder Paralichthys olivaceus and Analysis of Lactococcus garvieae Antigens by the Igs

“…Of these antigens, GAPDH has been reported as a vaccine candidate against Schistosoma mansonii and E. tarda (Argiro et al, 2000;Liu et al, 2005). In addition, PGK and OCT have been recognized as a protective antigen against various serotypes of Streptococcus agalactiae (Hughes et al 2002). AD is a putative virulence factor observed on the capsular surface of S. suis and S. pyogenes (Winterhoff et al 2002).…”

Extracellular virulence factors of group B Streptococci

“…Novel sophisticated molecular analyses such as signature-tagged mutagenesis (86), phage-display (63) or proteomics (87) are revealing further candidate GBS virulence determinants.…”

Vaccine efficacy of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Edwardsiella ictaluri against E. tarda in tilapia

“…Additionally, GAPDH is a common enzyme that exists in many organisms (Liu et al, 2005). Recent investigations of cell surface proteins of pathogenic streptococci and Gram-negative bacteria identified a cellwall-associated protein as structurally and functionally belonging to the GAPDH family of enzymes (Brassard et al, 2004;Hughes et al, 2002;Liu et al, 2005Liu et al, , 2007Sirover, 1999;Zhang et al, 2007). These studies demonstrated that GAPDH is well conserved among species.…”