Cleavage of Antigen-Bound Immunoglobulin G by SpeB Contributes to Streptococcal Persistence in Opsonizing Blood
Department of Biomedical Laboratory of Science,1 Department of Clinical Bacteriology, Umeå University, S-901 85 Umeå, Sweden2[m, http://www.100md.com
Received 4 March 2002/ Returned for modification 10 June 2002/ Accepted 23 September 2002[m, http://www.100md.com
ABSTRACT[m, http://www.100md.com
Group A streptococci (GAS) express a superantigen, SpeB, having cysteine protease activity. SpeB exhibits several properties that might contribute to virulence, the most recently discovered being the ability to cleave immunoglobulin G (IgG) in a manner similar to that of papain. In the present study, we confirmed this latter finding and found that the irreversible inhibition of SpeB protease activity completely abolishes IgG cleavage. SpeB cleavage of IgG was not species restricted since SpeB cleaved both human, rabbit, and mouse IgG. In order to investigate the nature of the SpeB cleavage of IgG, antibodies were immobilized prior to exposure to SpeB, either by unspecific binding of the Fc to GAS surface proteins or by antigen-specific binding. Analysis of the IgG molecules by SDS-PAGE showed that SpeB could cleave antigen-bound antibodies, while the IgG bound to IgG-binding proteins was protected from cleavage. In a phagocytosis assay using whole blood, the M49 GAS strain NZ131 showed a significantly higher survival than its isogenic speB mutant. Furthermore, the addition of extracellular supernatant derived from an overnight culture of native NZ131 increased the survival of its isogenic speB derivative. This indicates that SpeB's ability to cleave off the Fc part of antigen-bound IgG contributes to GAS escape from opsonophagocytosis while not interfering with the formation of a host-like coat by unspecific IgG binding.
INTRODUCTIONs8, http://www.100md.com
The group A streptococcus (GAS) is one of the most common human pathogens to cause a broad spectrum of diseases, ranging from mild infections to severe invasive diseases like necrotizing fasciitis and streptococcal toxic shock syndrome (42). Despite the hostile environment of the blood, GAS has the ability not only to survive but to grow and multiply in nonopsonizing blood. The underlying mechanisms suggested are the ability to avoid recognition by the immune system and interference with complement activation. M proteins are one of the virulence factors thought to be involved, since mutants lacking these surface proteins have a decreased capacity to escape phagocytosis. However, the underlying mechanisms are not fully understood and M proteins are reported to exhibit several functions that might be of importance. M proteins bind C4BP and factor H, which inhibits activation of the complement system through the alternative pathway, while binding of the Fc part of immunoglobulin G (IgG) by M proteins inhibits activation through the classical pathway (6, 23, 25, 29). The M and M-like surface proteins bind several plasma proteins, including fibronectin, albumin, plasminogen, and the Fc part of IgG and IgA, thereby covering the bacteria with host proteins (1, 7, 18, 20, 38). Production of a hyaluronic capsule as a coat is another way by which GAS avoid the action of the immune system (3, 36, 41). Other proteins, like the streptococcal inhibitor of complement and the C5a peptidase present at the GAS surface, further interfere with the activation of the complement system and recruitment of immune cells (2, 13). Nevertheless, opsonizing antibodies specific for M and M-like proteins are believed to be protective against GAS infections and confer long-term protection (30).
During infection, GAS secretes a number of soluble proteins, including those highly potent immune modulators the streptococcal pyrogenic exotoxins (Spe's), which are also recognized as streptococcal superantigens (33). The streptococcal superantigen SpeB is a cysteine protease with a wide variety of functions (19, 27, 28, 34). SpeB is produced as an inactive 40-kDa proenzyme that undergoes autocatalytic cleavage to the active 28-kDa form. The crystal structure of the 40-kDa zymogen reveals that SpeB belongs to the papain family (26). The proteolytic activity can be irreversibly inhibited by the addition of a tripeptide that covalently binds the active site, but inhibition of SpeB's proteolytic activity has no effect on its T-cell mitogenicity (8, 17).8y/5(, http://www.100md.com
Secretion of SpeB induces a number of events, creating an altered bacterial surface. It has been shown that inactivation of speB affects the expression of the hyaluronic capsule (4, 41). SpeB releases surface-associated C5a peptidase, which blocks leukocyte migration towards the site of infection induced by the chemotactic peptide C5a and degrades both the streptococcal inhibitor of complement and serum opacity factor (5, 21, 35). Furthermore, SpeB cleaves protein H and parts of M proteins from the surface of GAS, thus inhibiting the binding of fibronectin and altering the IgG-binding specificity (6, 10, 37). IgG bound to membrane-associated protein H does not activate the complement system, while soluble IgG-protein H complexes do, suggesting a mechanism to avoid activation of the complement system at the surface of the bacteria (6). In addition, SpeB was recently shown to cleave the heavy chain of human IgG (14).8y/5(, http://www.100md.com
Phagocytosis can be triggered through two different pathways, either via complement receptor 3 (CR3), also referred to as CD11b/CD18, or via the Fc(Anna Eriksson and Mari Norgren)
Received 4 March 2002/ Returned for modification 10 June 2002/ Accepted 23 September 2002[m, http://www.100md.com
ABSTRACT[m, http://www.100md.com
Group A streptococci (GAS) express a superantigen, SpeB, having cysteine protease activity. SpeB exhibits several properties that might contribute to virulence, the most recently discovered being the ability to cleave immunoglobulin G (IgG) in a manner similar to that of papain. In the present study, we confirmed this latter finding and found that the irreversible inhibition of SpeB protease activity completely abolishes IgG cleavage. SpeB cleavage of IgG was not species restricted since SpeB cleaved both human, rabbit, and mouse IgG. In order to investigate the nature of the SpeB cleavage of IgG, antibodies were immobilized prior to exposure to SpeB, either by unspecific binding of the Fc to GAS surface proteins or by antigen-specific binding. Analysis of the IgG molecules by SDS-PAGE showed that SpeB could cleave antigen-bound antibodies, while the IgG bound to IgG-binding proteins was protected from cleavage. In a phagocytosis assay using whole blood, the M49 GAS strain NZ131 showed a significantly higher survival than its isogenic speB mutant. Furthermore, the addition of extracellular supernatant derived from an overnight culture of native NZ131 increased the survival of its isogenic speB derivative. This indicates that SpeB's ability to cleave off the Fc part of antigen-bound IgG contributes to GAS escape from opsonophagocytosis while not interfering with the formation of a host-like coat by unspecific IgG binding.
INTRODUCTIONs8, http://www.100md.com
The group A streptococcus (GAS) is one of the most common human pathogens to cause a broad spectrum of diseases, ranging from mild infections to severe invasive diseases like necrotizing fasciitis and streptococcal toxic shock syndrome (42). Despite the hostile environment of the blood, GAS has the ability not only to survive but to grow and multiply in nonopsonizing blood. The underlying mechanisms suggested are the ability to avoid recognition by the immune system and interference with complement activation. M proteins are one of the virulence factors thought to be involved, since mutants lacking these surface proteins have a decreased capacity to escape phagocytosis. However, the underlying mechanisms are not fully understood and M proteins are reported to exhibit several functions that might be of importance. M proteins bind C4BP and factor H, which inhibits activation of the complement system through the alternative pathway, while binding of the Fc part of immunoglobulin G (IgG) by M proteins inhibits activation through the classical pathway (6, 23, 25, 29). The M and M-like surface proteins bind several plasma proteins, including fibronectin, albumin, plasminogen, and the Fc part of IgG and IgA, thereby covering the bacteria with host proteins (1, 7, 18, 20, 38). Production of a hyaluronic capsule as a coat is another way by which GAS avoid the action of the immune system (3, 36, 41). Other proteins, like the streptococcal inhibitor of complement and the C5a peptidase present at the GAS surface, further interfere with the activation of the complement system and recruitment of immune cells (2, 13). Nevertheless, opsonizing antibodies specific for M and M-like proteins are believed to be protective against GAS infections and confer long-term protection (30).
During infection, GAS secretes a number of soluble proteins, including those highly potent immune modulators the streptococcal pyrogenic exotoxins (Spe's), which are also recognized as streptococcal superantigens (33). The streptococcal superantigen SpeB is a cysteine protease with a wide variety of functions (19, 27, 28, 34). SpeB is produced as an inactive 40-kDa proenzyme that undergoes autocatalytic cleavage to the active 28-kDa form. The crystal structure of the 40-kDa zymogen reveals that SpeB belongs to the papain family (26). The proteolytic activity can be irreversibly inhibited by the addition of a tripeptide that covalently binds the active site, but inhibition of SpeB's proteolytic activity has no effect on its T-cell mitogenicity (8, 17).8y/5(, http://www.100md.com
Secretion of SpeB induces a number of events, creating an altered bacterial surface. It has been shown that inactivation of speB affects the expression of the hyaluronic capsule (4, 41). SpeB releases surface-associated C5a peptidase, which blocks leukocyte migration towards the site of infection induced by the chemotactic peptide C5a and degrades both the streptococcal inhibitor of complement and serum opacity factor (5, 21, 35). Furthermore, SpeB cleaves protein H and parts of M proteins from the surface of GAS, thus inhibiting the binding of fibronectin and altering the IgG-binding specificity (6, 10, 37). IgG bound to membrane-associated protein H does not activate the complement system, while soluble IgG-protein H complexes do, suggesting a mechanism to avoid activation of the complement system at the surface of the bacteria (6). In addition, SpeB was recently shown to cleave the heavy chain of human IgG (14).8y/5(, http://www.100md.com
Phagocytosis can be triggered through two different pathways, either via complement receptor 3 (CR3), also referred to as CD11b/CD18, or via the Fc(Anna Eriksson and Mari Norgren)