The importance of the location of antibody binding on the M6 protein for opsonization and phagocytosis of group A M6 streptococci.

Abstract

The M protein of group A streptococci is a fibrous dimer of a helices arranged in a coiled coil extending^" 50 nm from the surface of these organisms (1, 2) .Due to its profound antiphagocytic effect, this molecule serves as the primary virulence factor for these streptococci (3) which are cleared from the infected host only after type-specific opsonic antibodies are produced to the M protein (4). mAbs to the native M6 protein and DNA hybridization studies on the M6 gene indicate that the N11 2 -terminal region of this molecule is variable among M serotypes, whereas the carboxy-terminal half is conserved (5-8) . This suggests that type specificity may reside in the NH2-terminal half of the M protein, which is distal to the cell surface (9) . While a number of studies on a variety of M protein fragments derived from the NH 2-terminal half of the molecule suggest the importance of this region in the production of opsonic antibodies (10-14), no systematic approach concerning the binding site and function of specific antibodies to the complete M molecule has yet been reported .To help delineate the location of opsonogenic determinants on the M6 protein, mAbs raised against the native M6 molecule were examined for their ability to opsonize type 6 streptococci . Four of these monoclonals that had defined epitopes (reference 6 ; Jones, K . F ., S . K . Hollingshead, J . R . Scott, and V . A . Fischetti, manuscript in preparation) were examined for their IgG subclasses and ability to fix human complement . In addition, antisera against synthetic peptides representing various areas of the native M6 protein were examined for their opsonic capabilities . These studies revealed that opsonization of streptococci is a function of the location of the antibody-binding site on the M molecule . Materials and MethodsPurified M6 Proteins. M6 protein extracted from streptococcal cells by group C streptococcal phage-associated lysin (LysM6) was purified as described (15). M6 protein (ColiM6) was extracted and purified from an Escherichia coli strain expressing the structural gene for M6 protein (9) .Production of mAbs. mAbs against either LysM6 or ColiM6 were produced and protein A purified from culture supernatants as described (5, 6) .Bacterial Strains. Group A streptococcal clinical isolates representing 56 M serotypes were from The Rockefeller University culture collection and were listed previously (6) .This research was supported by a Public Health Service grant to V. A. Fischetti (AI-11822) . 1114J. Exp. Men.

New protective antigen of group A streptococci

“…In addition, M proteins elicit bactericidal antibodies thought to protect the host against subsequent infection by the same serotype (2). The antibodies with the greatest bactericidal activity are directed against the NH 2 -terminal, hypervariable epitopes of M proteins (21)(22)(23)(24). Among the many defined surface proteins of group A streptococci, only antibodies against the M protein have been shown to be opsonic.…”

Fibrinogen acts as a bridging molecule in the adherence of Staphylococcus aureus to cultured human endothelial cells.

“…Briefly, 10 mg of human fibrinogen (Sigma Chemical Co.) dissolved in PBS was added to 1 g of glutardialdehyde activated beads (Boehringer Mannheim Biochemicals). After incubation overnight at 4VC, the beads were washed sequentially with 1.5% NaCl, 0.3 M ethanolamine, 0.9% NaCl, 0.5 M propionic acid, 0.9% NaCl, and PBS, as described (29). Goat anti-human fibrinogen IgG (50 mg in 3 ml of PBS [Cappel Laboratories, Cochranville, PA]) was adsorbed to the fibrinogen-linked affinity column by rotating the mixture at 4VC overnight (8).…”

Identity and Prevalence of Multilocus Sequence Typing-Defined Clones of Group A Streptococci within a Hospital Setting

“…specific protection (23,28), necessitating the construction of multivalent vaccines by the use of hybrid proteins containing epitopes from several different M serotypes.…”