Cysteine Protease Activity and Histamine Release from the Human Mast Cell Line HMC-1 Stimulated by Recombinant Streptococcal Pyrogenic Exotoxin B/Streptococcal Cysteine Protease

Abstract

We constructed the expression vector pSK-SCP containing the streptococcal exotoxin B gene (spe b) which expressed protease activity. We showed that the recombinant streptococcal pyogenic exotoxin B/streptococcal cysteine protease (rSPE B/SCP) was secreted into the culture supernatant of the transformant and retained its SCP activity, which was equivalent to or greater than that of the naturally occurring molecule. The secreted rSPE B/SCP induced histamine release and degranulation of the human mast cell line HMC-1. This study may contribute to the understanding of the pathogenic role of SPE B/SCP in streptococcal infection and streptococcal toxic shock syndrome.Streptococcal pyrogenic exotoxin B (SPE B), known as streptococcal cysteine protease (SPE B/SCP) is highly conserved among group A Streptococcus (GAS). The structural gene, spe b, is found among all GAS clinical isolates (20) and expressed in almost all strains (12). This protein is initially secreted as a 42-kDa precursor, zymogen, and is autocatalytically cleaved to a 28-kDa mature SPE B/SCP (7, 9). SPE B/SCP cleaves or degrades fibronectin and vitronectin (12), which function as human matrix proteins. It also cleaves plasma kininogen and releases kinin (10) and cleaves the interleukin-1␤ (IL-1␤) precursor to the active form of IL-1␤ (12). Furthermore, SPE B/SCP activates the human matrix metalloprotease that increases type IV collagen degradation (2). Finally, genetically inactivated SPE B/SCP is attenuated in the mouse lethality model (15, 16). These findings suggest that SPE B/SCP activates inflammatory responses in the host and plays a very important role in pathogenesis of infections.The SPE B/SCP gene was previously cloned, sequenced (1, 9, 18), and expressed in Escherichia coli (6,8,17,19). However, the secretion of the active form of the protease from E. coli and its activity relative to the native molecule have not been examined. In order to clarify this question, we constructed an expression vector including spe b and its predicted promoter regions for transformation into E. coli. As a result, recombinant SPE B/SCP (rSPE B/SCP), retaining cysteine protease activity, was obtained from the culture supernatant.GAS strain NZ131 (kindly provided by D. R. Martin, NewZealand Communicable Disease Center, Porirua) chromosomal DNA was used as the template. Two oligonucleotides (SPEBF0008, 5ЈGTGTCAACTAACCGTGTTATTG-3Ј; SPEBR1485, 5Ј-TGATCTGTGTCTGASTGGATACTT-3Ј) were designed based on the spe b sequence as reported by Hauser et al. (9) and used as primers. PCR was performed with 25 cycles (94°C for 30 s, 54°C for 30 s, and 75°C for 1.5 min) and pyrobest DNA polymerase (TaKaRa Biomedicals, Kyoto, Japan). In order to construct the expression vector pSK-SCP, a 1,469-bp fragment including spe b and its predicted promoter region was amplified and cloned via the SmaI site in pBluescript II SK(ϩ) plasmid vector. pSK-SCP was transformed into E. coli strain JM109. The rSPE B/SCP was induced with 2 mM isopropyl-␤-D-thiogalactopyranoside (IPTG; Wako Pure Chemical Co...

Cysteine proteinase SpeB from Streptococcus pyogenes – a potent modifier of immunologically important host and bacterial proteins

“…SpeBm hydrolysis of H-kininogen in vitro and in vivo leads to release of the nona-peptide bradykinin, a potent vasodilator and inducer of pain, which most likely contributes to the pathophysiology of invasive S. pyogenes infections (Herwald et al , 1996 ). Another proinfl ammatory activity described for SpeBm is the induction of histamine release and degranulation of mast cells (Watanabe et al , 2002 ). The mechanism for the activity on mast cells is still unclear, but it seems to be coupled to SpeBm proteinase activity and could be a contributing factor to STSS (Navarre and Schneewind , 1999 ).…”

Contribution of CsrR-Regulated Virulence Factors to the Progress and Outcome of Murine Skin Infections byStreptococcus pyogenes

“…SpeB has a variety of mammalian substrates for its proteolytic activity, including interleukin-1␤ precursor, vitronectin, fibronectin, laminin, urokinase plasminogen receptor, and kininogens (14,18,20,36). In addition, this molecule induces mast cell degranulation (35) and activates the 66-kDa human endothelial cell matrix metalloprotease and has therefore been associated with endothelial cell damage (5). The potential degradation of a large variety of streptococcal exoproteins and M protein by SpeB may also influence virulence (19,22).…”

Extracellular Enzymes with Immunomodulating Activities: Variations on a Theme inStreptococcus pyogenes

“…Furthermore, SpeB directly cleaves and inactivates the antibacterial peptide LL-37, which is capable of killing S. pyogenes (96). Moreover, a recent study showed that purified SpeB stimulates the release of histamine from a human mast cell line (113). Some reports have suggested that SpeB also functions as a superantigen, with stimulation of T lymphocytes without antigen presentation, and that the activity is independent of the proteolytic activity (30,64).…”