Ａ群鏈球菌(Streptococcus pyogenes)的感染會引起人類許多種疾病，臨床上所產生嚴重的併發症包括：鏈球菌毒性休克症候群(streptococcal toxin shock syndrome)和壞疽性肌膜炎(necrotizing fascilitis)。熱原性外毒素B (SPEＢ)的基因存在所有的Ａ群鏈球菌中，在Ａ群鏈球菌的感染上扮演重要的角色。SPE B在功能上屬於cysteine protease，原以40 kDa的zymogen形式存在，會進一步活化成 28 kDa的成熟型SPE B。

目前在已知許多感染中，細胞凋亡（apoptosis）是細菌的重要致病機轉之一。而在我們實驗室過去的研究指出， SPE B可引發人類呼吸道上皮細胞株 (A549) ; 而由SPE B引發的A549細胞凋亡可能是經由receptor所傳導。故我們利用體外細胞培養模式，以U937來探討SPE B在引發細胞凋亡中所扮演的角色。以SPE B和其點突變株C192S（28 kDa，不具有酵素活性）處理U937細胞，結果皆有細胞凋亡的情形，但SPE B所造成的細胞凋亡程度較28 kDa C192S來的高。由觀察28 kDa C192S所造成的細胞凋亡實驗結果顯示，U937在進行吞噬或胞飲作用的過程中，可能使得receptor顯現出來而造成部份的細胞凋亡。然而，當我們以低劑量的SPE B先處理U937細胞5、10及20 分鐘，接著再加入28 kDa C192S 作用24小時，結果顯示隨著先處理SPE B的時間增長，細胞凋亡的情形也越增加。由以上實驗結果可知不論是SPE B的大小或蛋白脢活性在引發細胞凋亡過程中皆扮演著重要的角色。此外當我們同時加入αvβ3抗體時，可阻止由SPE B引發的細胞凋亡，而另一方面的實驗指出G308S (SPE B在RGD motif的突變株)並不會引發U937細胞凋亡，故我們認為SPE B和αvβ3 integrin 的結合是引發U937細胞凋亡主要步驟。此外，在螢光免疫偵測細胞表面receptor實驗發現，以低劑量SPE B先處理細胞20分鐘，可偵測到較多的αvβ3 integrin，故我們推測SPE B的蛋白脢活性作用於細胞表面，和顯現出αvβ3 receptor有關。其他實驗發現，外加general caspase抑制劑、caspase-8抑制劑及caspase-3抑制劑，可阻礙SPE B引發的細胞凋亡; 而以SPE B處理細胞後，可以偵測到活化型的caspase-8、BAX、caspase-3和cytochrome c 由
粒線體釋出至細胞質。另一方面的實驗指出SPE B會降低U937細胞的吞噬能力，而E64 (蛋白脢抑制劑)的加入會阻斷SPE B的吞噬抑制作用。分別處理papain (cysteine 蛋白脢)及trypsin (serine蛋白脢)皆不會降低U937細胞的吞噬能力。由以上實驗結果可知SPE B的蛋白脢活性對SPE B引發的吞噬抑制作用是重要且具有專一性的。

Streptococcus pyogenes can cause serious diseases in humans, including life-threatening streptococcal toxin shock syndrome and necrotizing fascilitis. Streptococcal Pyrogenic Extoxin B (SPE B), a cysteine protease carried by every strain of S. pyogenes, may be a critical virulence factor in streptococcal infections. SPE B is synthesized as a 40kDa zymogen, and subsequently converted to a 28kDa active protease.
Apoptosis has been implicated in the mechanism of bacterial pathogenesis. In our lab previous studies found that purified rSPE B protein could induce apoptosis in A549 (human respiratory epithelial cells) cells, and suggested that the rSPE B-induced A549 apoptosis may be mediated by a receptor-like mechanism. In this study, we used an in vitro system to elucidate the mechanism of how SPE B induces apoptosis in U937 cells. Both purified rSPE B (28kDa) and mutant SPE B (28kDa C192S, which has no protease activity) were used for this study. Our results showed that both of them could induce U937 cells to undergo apoptosis and the rSPE B-induced apoptosis was more than that induced by mutant SPE B. The studies on 28 kDa C192S mutant -induced apoptosis suggested that phagocytosis might expose some binding site for rSPE B. However, when cells were pretreated with low concentration of rSPE B for short time (5, 10 and 20 min) and then incubated with 28kDa C192S mutant for 24 hrs, the extent of apoptosis was similar to that induced by rSPE B for 24hrs. These results indicated that the size of SPE B, and its protease activity are important for its function to induce apoptosis. In addition, when cells were incubated with αvβ3 antibody, the rSPE B-induced apoptosis was almost completely blocked, and G308S (mutant SPE B in RGD motif) did not induce U937 cell apoptosis, suggesting that the binding of SPE B to αvβ3 integrin was the important step for rSPE B-induced apoptosis. In consistent with these results, immunofluorescence labeling of surface receptor (αvβ3 integrin) showed that pretreatment of cells with low dose rSPE B for 20 min could expose more αvβ3 integrin. Treatment of cells with Z-IETD-FMK (caspase-8 inhibitor), Z-VAD-FMK (general caspase inhibitor) and Ac-DEVD-CHO (caspase-3 inhibitor) could inhibit the rSPE B-induced apoptosis. Our studies also showed that other factors; such as caspase 8, BAX, caspase 3 and cytochrome c release were activated by rSPE B in U937 cells. Other experiments showed that the phagocytosis activity of U937 cells was reduced by rSPE B. Treatment of cells with E64 (cysteine protease inhibitor) abrogated this phagocytosis- inhibitory effect by SPE B. The treatment with papain (cysteine protease) and trypsin (serine protease) could not reduce the phagocytosis activity in U937 cells. These results indicated that the protease of rSPE B is important and specific to rSPE B-reduced phagocytosis activity.

Bhakdi, S., Roth, M., Sziegoleit, A., and Tranum-Jensen, J. 1984. Isolation of two hemolytic forms of streptolysin O. Infect. Immun. 46:394-400.

Black, D. S. and Bliska, J. B. 1997. Identification of p130Cas as a substrate of Yersinia YopG, a bacterial protein tyrosine phosphatase that translocates into mammalian cells and targets focal adhesions. EMBO J. 16:2730-2744.

Burns, E. H., Marciel, Jr., A. M., and Musser, J. M. 1996. Activation of a 66 kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular cysteine protease. Infect. Immun. 64:4744-4750.

Caron, E., and A. Hall. 1998. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 282:1717-1721.

Chen Y., and A. Zychlinsky. 1994. Apoptosis induced by bacterial pathogens. Microb. Pathog. 17:203-212.

D’ Costa, S. S. and Boyle, M. D. 1998. Interaction of a group A streptococcus within human plasma results in assembly of a surface plasminogen acrtivator that contributes to occupancy of surface plasmin-binding structure. Microb. Pathog. 24: 341-349.

Ernst, J. D. 2000. Bacterial inhibition of phagocytosis. Cell. Microbiol. 2:379-386.

Finlay, B. B., and P. Cossart. 1997. Exploitation of mammalian host cell functions by bacterial pathogens. Science 276:718-725.

Guzman, C. A., E. Domann, M. Rohde, D. Bruder, A. Darji, S. Weiss, J. Wehland, T. Chakraborty, and K. N. Timmis. 1996. Apoptosis of mouse dendritic cells is triggered by listeriolysin, the major virulence determinant of L. monocytogenes. Mol. Microbiol. 20:119-126.

Herwald, H., Collin, M., Muller-Esterl, W., and Bjorck, L. 1996. Streptococcal cysteine protease releases kinins: a novel virulence mechanism. J. Exp. Med. 184:665-673.

Holm, S. E., Norrby, A., Gergholm, A-M., and Norgren, M. 1992. Aspects of pathogenesis of serious group A streptococcal infections in Sweden, 1988-1989. J. Infect. Dis. 166:31-37.

Hsueh P. R., J. J. Wu, P. J. Tsai, J. W. Liu, Y. L. Chuang and K. T. Luh. 1998. Invasive group A streptococcal disease in Taiwan is not associated with the presence of Streptococcal Pyrogenic Extoxin genes. Clinical infectious disease. 26:584-589.

Ji, Y., McLandsborough, L., Kondagunta, A., and Cleary, P. P. 1996. C5a peptidase alters clearance and trafficking of group A streptococci by infected mice. Infect. Immun. 64:503-510.

Kapur, V., Topouzis, S., Majeasky, M. W., Li, L. L., Hamrick, M. R., Hamill, R. J., Patti, J. M., and Musser, J. M. 1993a. A conserved streptococcus pyogenes extracellular cysteine protease cleaves human fibronectin and degrades vitronectin. Micob. Pathog. 15:327-346.

Kapur, V., Majeasky, M. W., Li, L. L., Black, R. A., and Musser, J. M. 1993b. Cleavage of interleukin 1β (IL-1β) precursor to produce active IL-1β by a conserved extracellular cysteine protease from Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA. 90:7676-7680.

Keane, J., M. K. Balcewicz-Sablinska, H. G. Remold, G. L. Chupp, B. B. Meek, M. J. Fenton, and H. Kornfeld. 1997. Infection by mycobacterium tuberculosis promotes human alveolar macrophage apoptosis. Infect. Immun. 65:298-304.

Kischdel F. C., S. Hellbardt, I. Behrmann, M. Germer, M. Pawlita, P. H. Krammer. 1995. cytotoxicity-dependent APO-I(Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 14:5579-5588.

Kuo, C. F., J. J. Wu, P.J. Tsai, P. J. Lei, M. T. Lin, and Y. S. Lin. 1999. Streptococcal pyrogenic exotoxin B induces apoptosis and reduces phagocytic activity in U937 cells. Infect. Immun. 67:126-130.

Kuo, C. F., J. J. Wu, P.J. Tsai, S. C. Lee, U. T. Jin, H. Y. Lei, and Y. S. Lin. 1998. Role of streptococcal pyrogenic exotoxin B in the mouse model of group A streptococcal infection. Infect. Immun. 66:3931-3935.

Li H., Zhu H., Xu C. and Yuan J. 1998. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94:491-501.

Liles, W. C. 1997. Apoptosis: role in infection and inflammation. Curr. Opin. Infect. Dis. 10:165-170.

Liu X., Kim C. N., Yang J., Jemmerson R. and Wang X. 1996. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86:147-157.

Lukomski, S., Sreevatsan, S., Amgereg, C., Reichardt, W., Woischnik, M., Podbielski, A., and Musser, J. M. 1997. Inactivation of streptococcus pyogenes extracellular cysteine protease significantly decreases mouse lethality of serotype M3 and M49 strains. J. Clin. Invest. 99:2574-2580.

Lukomski, S., E. H. Burns, Jr., P. R. Wyde, A. Podbielski, J. Rurangirwa, D. K. Moore-Poveda, and J. M. Musser. 1998. Genetic inactivation of an extracellular cysteine protease (spe B) expressed by streptococcus pyrogenes decreases resistance to phagocytosis and dissemination to organs. Infect. Immun. 66:771-776.

Luo X., Budihardjo I., Zou H., Slaughter C. and Wang X. 1998. Bid, a Bcl-2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94:481-490.

Mason, P. W., Rieder, E. and Baxt, B. 1994. Proc.Natl. Acad. Sci. USA. 91:1932-1936.

Monack, D. M., B. Raupach, A. E. Hromockyj, and S. Falkow. 1996. Salmonella typhimurium invasion induces apoptosis in infected macrophages. Proc. Natl. Acad. Sci. USA 93:9833- 9838.

Muller, A., J. Hacker, and B. C. Brand. 1996. Evidence for apoptosis of human macrophage-like HL-60 cells by Legionella pneumophila infection. Infect. Immu. 64:4900-4906.

Musser, J.M., Hauser, A. R., Kim, M. H.., Schlievert, P. M., Nelson, K., and Selander, R. K. 1991. Streptococcus pyogenes causing toxic-shock-like syndrome and other invasive disease: clonal diversity and Pyrogenic exotoxin expression. Proc. Natl. Acad. Sci. USA 88: 2668 –2672.

Musser, J.M., and Krause, R. M. 1998.The revival of group A streptococcal diseases, with a commentary on staphylococcal toxic shock syndrome. In Emerging Infections. R. M. Krause, editor. Academic Press. New York, New York, USA. 185-218.

Musser, J.M., Stockbauer, K., Kapur, V., and Rudgers, G. W. 1996. Substitution of cysteine 192 in a highly conserved streptococcus pyrogenes extracellular cysteine protease (interleukin 1β convertase) alters proteolytic activity and ablates zymogen processing. Infect. Immun. 64: 1913-1917.

Norrby-Teglund A., Norgren, M., Holm, S. E., Aaanderson, U., and Anderson, J. 1994. Similar cytokine induction profiles of a novel streptococcal exotoxin, MF, and yrogenic axotoxins A and B. Infect. Immun. 62: 3731-3738.

Ohara-Nemoto, Y., Sasaki, M., Kanedo, M., Nemoto, T., and Ota, M. 1994. Cysteine protease activity of streptococcal pyrogenic exotoxin B. Can. J. Microbiol. 40: 930-936.

O’Connor, S. P. and Cleary, P. P.1986. Localization of the streptococcal C5a peptidase to the surface of group A streptococci. Infect. Immun. 53: 432-434.

Raeder, R. and Boyel, M. 1993. Association between expression of immunoglobulin G-binding proteins by group A streptococci and virulence in a mouse skin infection model. Infect. Immun. 61:1378-1384.

Rachel, N. 1994. Flesh-eating bacteria: not new, but still worrisome. Science 264:1665.

Relman, D. A., Domenighini, M., Tuomanen, E., Rappuoli, R. and Falkow, S. 1989. Proc. Natl. Acad. Sci. USA. 86:2637-2641.

Robinson, J. H. and Kehoe, M. A. 1992. Group A streptococcal M proteins: vilulence factors and protective antigens. Immunol. Today 13:362-367.

Ruckdeschel, K., A. Roggenkamp, V. Lafont, P. Mangeat, J. Heesemann, and B. Rouot. 1997. Interaction of Yersinia enterocolitica with macrophages leads to macrophage cell death through apoptosis. Infect. Immun. 65:4813-4821.

Stevens, D. L., Tanner, M. H., and Winship, J. 1989. Sever group A streptococcal infections associated with a toxic shock like syndrome and scarlet fever toxin A. N. Eng. J. Med. 321:1-8.

Stevens, D. L. 1997. The toxins of group A streptococcus, the flesh eating bacteria. Immun. Investigations 26:129-150.

Stockbauer, K.E. 1999. A natural variant of the cysteine protease virulence factor of group A Streptococcus with an RGD motif preferentially binds human integrins αvβ3 and αIIbβ3. Proc. Natl. Acad. Sci. USA. 96:242-247.

Tsai, P-J., Kuo, C-H., Lin, K-Y., Lei, H-Y., Chen, F-F., Wang, J-R., and Wu, J-J. 1998. Effect of group A streptococcal cysteine protease on invasion of epithelial cells. Infect. Immun. 66: 1460-1466.

Vaux, D. L. and Strasser, A. 1996. The molecular biology of apoptosis. Proc. Natl Acad. Sci. USA 83:2239-2244.

Van Oss, C. R. 1986. Phagocytosis: an overview. Methods Enzymol. 192:3-15.

Wessels, M. R., Moses, A. E., Goldberg, J. B. and DiCesare, T. J. 1991. Hyaluronic acid capsule is a virulence factor for mucoid group A streptococci. Proc. Natl. Acad. Sci. USA 88:8317-8321.

White, E. 1996. Life, death and the pursuit of apoptosis. Genes. Dev. 10:1-15.

Yonaha, K., S. D. Elliott, and T. Y. Liu. 1982. Primary structure of zymogen of Streptococcal proteinase. J. Protein Chem. 1:317-334.

Yu., C. E., and J. J. Ferretti. 1991.Frequence of the erythromycin toxin B and C genes (speB and speC) among clinical isolates of group A streptococci. Infect. Immun. 59: 211-215.

Zychlinsky, A., M. C. Prevost, and P. J. Sansonetti. 1992. Shigella flexneri induces apoptosis in infected macrophages. Nature 358:167-168.

Zhou D. G., Mooseker M. S., Galan J. E. 1999. An invasion-associated Salmonella protein modulates the actin-bundling activity of plastin. Proc. Natl. Acad. Sci. USA 96:10176-10181.

郭志峰 1999. Role of Streptococcal Pyrogenic Exotoxin B in Streptococcus pyogenes Infection. 國立成功大學基礎醫學研究所博士論文.

蔡宛樺 2000. Studies on the Streptococcal Pyrogenic Exotoxin B-induced Apoptosis in A549 cell. 國立成功大學生物化學研究所碩士論文.