Proapoptotic effect of proteolytic activation of matrix metalloproteinases by Streptococcus pyogenes thiol proteinase (Streptococcus pyrogenic exotoxin B).
Streptococcus pyogenes thiol proteinase, also known as streptococcal pyrogenic exotoxin B (SpeB), has been suggested to be a major virulence factor in S. pyogenes infection. SpeB was reported to induce apoptosis of host cells, but its mechanism of action is not yet fully understood. In this study, we examined the involvement of matrix metalloproteinases (MMPs) in SpeB-induced apoptosis. We first developed a large-scale preparation of recombinant SpeB and precursors of human MMP-9 and -2 (proMMPs) by using Escherichia coli Rosetta (DE3)pLysS and baculovirus-insect cell expression systems, respectively. Treatment with SpeB induced effective proteolytic activation of both proMMP-9 and -2. When RAW264 murine macrophages were incubated with SpeB-activated proMMP-9, the level of tumor necrosis factor alpha (TNF-alpha) in conditioned medium (CM), assessed by an enzyme immunoassay, was elevated. This increase was completely inhibited by addition of the MMP inhibitor SI-27 to the cell culture. The CM also produced marked induction of apoptosis of U937 human monocytic cells. Similarly, soluble Fas ligand (sFasL) was detected in CM of cultures of SW480 cells expressing FasL after treatment with SpeB-activated proMMPs; this CM also induced apoptosis in U937 cells. SpeB had a direct effect as well and caused the release of TNF-alpha and sFasL from the cells. SpeB-dependent production of MMP-9 and -2 and proapoptotic molecules (TNF-alpha and sFasL) was evident in a murine model of severe invasive S. pyogenes infection. These results suggest that SpeB or SpeB-activated MMPs contribute to tissue damage and streptococcal invasion in the host via extracellular release of TNF-alpha and sFasL.
Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan.
Fumio
F
Tamura
Rumiko
R
Nakagawa
Teruo
T
Akuta
Shigefumi
S
Okamoto
Shigeyuki
S
Hamada
Hiroshi
H
Maeda
Shigetada
S
Kawabata
Takaaki
T
Akaike
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D017364:Collagenases / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D005098:Exotoxins / Q000378:metabolism / Q000494:pharmacology*; D053222:Fas Ligand Protein; D018093:Gelatinases / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008264:Macrophages; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D013290:Streptococcal Infections / Q000382:microbiology / Q000503:physiopathology*; D013297:Streptococcus pyogenes / Q000472:pathogenicity*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020298:U937 Cells
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D005098:Exotoxins; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D014409:Tumor Necrosis Factor-alpha; C027951:erythrogenic toxin; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; C120487:pro-matrix metalloproteinase 9; C069860:progelatinase; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9