Group A Streptococcal Cysteine Protease Degrades C3 (C3b) and Contributes to Evasion of Innate Immunity

Abstract

A relative lack of neutrophils around Streptococcus pyogenes is observed in streptococcal toxic shock syndrome (STSS). Because the bacteria spread rapidly into various organs in STSS, we speculated that S. pyogenes is equipped with molecules to evade the host innate immune system. Complement C3b opsonizes the pathogen to facilitate phagocytosis, and a complex of C3b converts C5 into anaphylatoxin. Because we found that C3 (C3b) is degraded in sera from patients with STSS, we investigated the mechanism of C3 (C3b) degradation by S. pyogenes. We incubated human C3b or serum with recombinant SpeB (rSpeB), a wild-type S. pyogenes strain isolated from an STSS patient or its isogenic ⌬speB mutant and examined the supernatant by Western blotting with anti-human C3b. Western blot and Biacore analyses revealed that rSpeB and wild-type S. pyogenes rapidly degrade C3b. Additionally, C3 (C3b) was not detected in sera collected from infected areas of STSS patients. Furthermore, the survival rate in human blood and in mice was lower for the ⌬speB mutant than the wild-type strain. Histopathological observations demonstrated that neutrophils were recruited to and phagocytosed the ⌬speB mutant, whereas with the wild-type strain, few neutrophils migrated to the site of infection, and the bacteria spread along the fascia. We observed the degradation of C3 (C3b) in sera from STSS patients and the degradation of C3 (C3b) by rSpeB. This suggests that SpeB contributes to the escape of S. pyogenes from phagocytosis at the site of initial infection, allowing it to invade host tissues during severe infections.Streptococcus pyogenes is a Gram-positive bacterium that often causes throat and skin infections such as pharyngitis and impetigo. During the past decade, it was reported that S. pyogenes causes severe infectious diseases, including acute rheumatic fever, necrotizing fasciitis, and streptococcal toxic shock syndrome (STSS) 2 (1, 2). The death toll from severe infections was estimated to be at least 500,000 each year (2). Therefore, many researchers have focused on S. pyogenes, and some aspects of the infection have been elucidated.The initial step of S. pyogenes infection is bacterial adhesion to host epithelial cells through extracellular matrix proteins, for example, fibronectin (3-5). Fibronectin-binding proteins of S. pyogenes have been identified as adhesins and invasins, and their role in the invasion of epithelial cells by S. pyogenes has been examined in several studies (6, 7); however, how the invading bacteria escape the immune system and grow in host tissues is not understood. To cause systemic and septic infections, S. pyogenes must evade the immune system of the host and survive in plasma, after which the organisms may spread into various organs via the blood stream. Histopathological studies have demonstrated that there are few or no inflammatory cells (e.g. neutrophils) at the site of infection in patients with STSS and severe streptococcal infections (8, 9).The complement system plays an important role in innate immu...

Streptococcus pyogenes Endopeptidase O Contributes to Evasion from Complement-mediated Bacteriolysis via Binding to Human Complement Factor C1q

“…On the other hand, lack of neutrophil migration to sites of infection with S. pyogenes in the early phase of STSS has been shown (40,41). Notably, the SSI-9 strain was shown in previous studies to produce SpeB, which degrades C3b and enables evasion from opsonophagocytosis by neutrophils (41). Consequently, no pH decrease may occur in inflammatory tissues because of a lack of neutrophil migration.…”

“…Accordingly, accumulation of lactic acid by fermentation induces a significant decrease in pH level at the inflammatory locus (39). On the other hand, lack of neutrophil migration to sites of infection with S. pyogenes in the early phase of STSS has been shown (40,41). Notably, the SSI-9 strain was shown in previous studies to produce SpeB, which degrades C3b and enables evasion from opsonophagocytosis by neutrophils (41).…”

“…To examine PepO expression levels in the obtained samples, Western blot analysis was performed, as previously described, with minor modifications (41,58). Briefly, PepO in each sample was detected using mouse anti-PepO antiserum diluted to a concentration of 1:2000.…”

Neisseria meningitidis NalP cleaves human complement C3, facilitating degradation of C3b and survival in human serum

“…Unlike NalP, aureolysin cleaves C3 2 aa C-terminal to the C3 convertase cleavage site to generate functionally active C3b and C3a (28). In contrast to NalP, the streptococcal cysteine protease SpeB fully degrades C3 as a result of cleavage at multiple sites (25).…”

“…Proteolytic cleavage of complement components by bacterial proteases is a mechanism used by many human pathogens. Examples are streptococcal cysteine protease SpeB, which degrades C3 to inhibit bacterial clearance (25), the streptococcal cell-associated peptidase ScpA, which cleaves C5a to inhibit neutrophil chemotaxis (26), GelE of Enterococcus faecalis (27) and aureolysin of Staphylococcus aureus, which cleave C3 (28), EspP of enterohemorrhagic E. coli, which cleaves C3 and C5 to impair complement activation (29), and PgtE of Salmonella enterica, which proteolytically cleaves C3b, C4b, and C5 to enhance bacterial resistance to human serum (30).…”