化膿性鏈球菌 (Streptococcus pyogenes) 又稱為A群鏈球菌 (group A streptococcus) 是一種人類致病菌，可造成輕微的咽喉炎到嚴重的鏈球菌毒素休克症候群等多樣化疾病。在A群鏈球菌中，Peroxide stress response regulator (PerR) 為fur家族成員之一，同時也扮演維持金屬離子平衡及適應氧化壓力之調控子的角色。先前研究指出在動物感染模式中，perR突變株的毒力表現比野生株弱，顯示PerR可能調控某些適應環境相關的毒力因子。過去我們的團隊以Real-time PCR分析野生株與perR突變株在一般培養環境與氧化壓力環境下DNase基因 (sda1, mf3, spd) 的表現，發現野生株在氧化壓力下sda1基因表現量會增加，但perR突變株則不會。因此本論文進一步探討PerR是如何調控細菌噬菌體DNase基因－sda1。首先，我們以cDNA五端快速放大法找到sda1轉錄位點在轉譯位點上游第26個核苷酸位置。以回補野生型perR基因到perR突變株後，發現在氧化壓力環境下可以顯著地提升sda1基因表現，但回補鐵離子調控區突變的PerR H99A，則無；顯示PerR的金屬結合位點對於sda1的調控相當重要。此外，在過氧化氫刺激下，野生株sda1啟動子活性會被提升，但perR突變株則不會，這結果再次佐證當A群鏈球菌處在氧化壓力的環境下PerR可以調控sda1轉錄層次的表現。我們以電泳位移試驗進一步證實PerR能直接與sad1啟動子結合，且具專一性。同時在sda1啟動子-144~ -174與-22~-36找到兩個可能的PerR結合位。這些結果顯示A群鏈球菌在受到氧化壓力的刺激後，PerR具有正向調控sda1表現的能力。此研究對於A群鏈球菌PerR在氧化壓力下調控毒力因子提供重要的證據。

Streptococcus pyogenes (group A streptococcus) is a human pathogen, which can cause various diseases from mild pharyngitis to severe streptococcal toxic shock syndrome. Peroxide stress response regulator (PerR) is the member of ferric uptake repressor (fur) super-family, and is the transcriptional regulator for metal homeostasis and oxidative stress response in S. pyogenes. Previous studies showed that the virulence of perR mutant was attenuated than the wild-type in animal in ection models, and it suggested PerR may regulate virulence factors to adapt the environmental changes. Moreover, our lab analyzed DNase genes (sda1, mf3, spd) expression in the wild-type strain and the perR mutant under normal culture conditions and oxidative stress conditions by Real-time PCR, and found that sda1 expression in the wild-type strain was increased under oxidative stress, but perR mutant did not. The aim of this thesis was to demonstrate how PerR regulate bacteriophage-encoded gene, sda1. First, the rapid amplification of 5’ cDNA ends assay demonstrated that the transcriptional start site of sda1 was located at the 26 nucledtides upstream of the translational start site. PerR and PerR H99A mutation, the Fe2+ binding site of PerR, were complemented in the perR mutant to observe the sda1 expression. The results showed that the sda1 expression was increased under hydroxy peroxide treatment in complemented PerR but not in complemented PerR H99A. These results suggested that the metal binding site was important for PerR to regulate sda1. In addition, the sda1 promoter ability was increased under oxidative stress in the wild-type stain but not in the perR mutant. Further, we found PerR can specifically bind to the sda1 promoter by the electrophoretic mobility shift assay. The two Per boxes were found at sda1 promoter -144~ -174 and -22~-36 regions. Our data suggested that PerR can positively regulate sda1 under oxidative stress conditions. This study provides evidence to support the PerR regulates the virulence factors under oxidative stress in group A streptococcus.

Anbalagan, S., and Chaussee, M. S. 2013. Transcriptional regulation of a bacteriophage encoded extracellular DNase (Spd-3) by Rgg in Streptococcus pyogenes. PLoS One 8:e61312.
Aziz, R. K., Ismail, S. A., Park, H. W., and Kotb, M. 2004. Post-proteomic identification of a novel phage-encoded streptodornase, Sda1, in invasive M1T1 Streptococcus pyogenes. Mol Microbiol 54:184-97.
Aziz, R. K., Pabst, M. J., Jeng, A., Kansal, R., Low, D. E., Nizet, V., and Kotb, M. 2004. Invasive M1T1 group A streptococcus undergoes a phase-shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB. Mol Microbiol 51:123-34.
Beall, B., Facklam, R., and Thompson, T. 1996. Sequencing emm-specific PCR products for routine and accurate typing of group A streptococci. J. Clin. Microbiol. 34:953-58.
Berge, A., and Sjöbring, U. 1993. PAM, a novel plasminogen-binding protein from Streptococcus pyogenes. J Biol Chem 268:25417-24.
Brenot, A., King, K. Y., and Caparon, M. G. 2005. The PerR regulon in peroxide resistance and virulence of Streptococcus pyogenes. Mol Microbiol 55:221-34.
Buchanan, J. T., Simpson, A. J., Aziz, R. K., Liu, G. Y., Kristian, S. A., Kotb, M., Feramisco, J., and Nizet, V. 2006. DNase expression allows the pathogen group A streptococcus to escape killing in neutrophil extracellular traps. Curr Biol 16:396-400.
Carapetis, J. R., Steer, A. C., Mulholland, E. K., and Weber, M. 2005. The global burden of group A streptococcal diseases. Lancet Infect Dis 5:685-94.
Chan, J. C., Chu, Y. W., Chu, M. Y., Cheung, T. K., and Lo, J. Y. 2009. Epidemiological analysis of Streptococcus pyogenes infections in Hong Kong. Pathology 41:681-6.
Chiang-Ni, C., Wu, A. B., Liu, C. C., Chen, K. T., Lin, Y. S., Chuang, W. J., Fang, H. Y., and Wu, J. J. 2011. Emergence of uncommon emm types of Streptococcus pyogenes among adult patients in southern Taiwan. J Microbiol Immunol Infect 44:424-9.
Chiou, C. S., Wang, Y. W., Chen, P. L., Wang, W. L., Wu, P. F., and Wei, H. L. 2009. Association of the shuffling of Streptococcus pyogenes clones and the fluctuation of scarlet fever cases between 2000 and 2006 in central Taiwan. BMC Microbiol 9:115.
Cole, J. N., Barnett, T. C., Nizet, V., and Walker, M. J. 2011. Molecular insight into invasive group A streptococcal disease. Nat Rev Microbiol 9:724-36.
Cole, J. N., Pence, M. A., von Kockritz-Blickwede, M., Hollands, A., Gallo, R. L., Walker, M. J., and Nizet, V. 2010. M protein and hyaluronic acid capsule are essential for in vivo selection of covRS mutations characteristic of invasive serotype M1T1 group A streptococcus. MBio 1:e00191-10.
Courtney, H. S., Hasty, D. L., and Dale, J. B. 2002. Molecular mechanisms of adhesion, colonization, and invasion of group A streptococci. Ann Med 34:77-87.
Cunningham, M. W. 2000. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 13:470-511.
Daniel, S., and Ferretti, J. J. 1991. Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA. FEMS Microbiol Lett 82:219-24.
Dubbs, J. M., and Mongkolsuk, S. 2012. Peroxide-sensing transcriptional regulators in bacteria. J Bacteriol 194:5495-503.
Fuangthong, M., and Helmann, J. D. 2003. Recognition of DNA by three ferric uptake regulator (Fur) homologs in Bacillus subtilis. J Bacteriol 185:6348-57.
Gibson, C. M., and Caparon, M. G. 1996. Insertional inactivation of Streptococcus pyogenes sod suggests that prtF is regulated in response to a superoxide signal. J Bacteriol 178:4688-95.
Goshorn, S. C., and Schlievert, P. M. 1989. Bacteriophage association of streptococcal pyrogenic exotoxin type C. J Bacteriol 171:3068-73.
Gryllos, I., Grifantini, R., Colaprico, A., Cary, M. E., Hakansson, A., Carey, D. W., Suarez-Chavez, M., Kalish, L. A., Mitchell, P. D., White, G. L., and Wessels, M. R. 2008. PerR confers phagocytic killing resistance and allows pharyngeal colonization by group A streptococcus. PLoS Pathog 4:e1000145.
Hakansson, A., Bentley, C. C., Shakhnovic, E. A., and Wessels, M. R. 2005. Cytolysin-dependent evasion of lysosomal killing. Proc Natl Acad Sci U S A 102:5192-7.
Hasegawa, T., Minami, M., Okamoto, A., Tatsuno, I., Isaka, M., and Ohta, M. 2010. Characterization of a virulence-associated and cell-wall-located DNase of Streptococcus pyogenes. Microbiology 156:184-90.
Hasegawa, T., Torii, K., Hashikawa, S., Iinuma, Y., and Ohta, M. 2002. Cloning and characterization of two novel DNases from Streptococcus pyogenes. Arch Microbiol 177:451-6.
Hasty, D. L., Ofek, I., Courtney, H. S., and Doyle, R. J. 1992. Multiple adhesins of streptococci. Infect Immun 60:2147-52.
Hauser, A. R., and Schlievert, P. M. 1990. Nucleotide-sequence of the streptococcal pyrogenic exotoxin type-b gene and relationship between the toxin and the streptococcal proteinase precursor. J Bacteriol 172:4536-42.
Hayashi, K., Ohsawa, T., Kobayashi, K., Ogasawara, N., and Ogura, M. 2005. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in Bacillus subtilis. J Bacteriol 187:6659-67.
Hellman, L. M., and Fried, M. G. 2007. Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc 2:1849-61.
Imlay, J. A. 2008. Cellular defenses against superoxide and hydrogen peroxide. Annu Rev Biochem 77:755.
Ish-Horowicz, D., and Burke, J. 1981. Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2989-2898.
Iwasaki, M., Igarashi, H., and Yutsudo, T. 1997. Mitogenic factor secreted by Streptococcus pyogenes is a heat-stable nuclease requiring His122 for activity. Microbiology 143:2449-55.
King, K. Y., Horenstein, J. A., and Caparon, M. G. 2000. Aerotolerance and peroxide resistance in peroxidase and PerR mutants of Streptococcus pyogenes. J Bacteriol 182:5290-9.
Kotb, M. 1995. Bacterial pyrogenic exotoxins as superantigens. Clin Microbiol Rev 8:411-26.
Kreikemeyer, B., Klenk, M., and Podbielski, A. 2004. The intracellular status of Streptococcus pyogenes: role of extracellular matrix-binding proteins and their regulation. Int J Med Microbiol 294:177-88.
Lancefield, R. C. 1933. A serological differentiation of human and other groups of hemolytic streptococci. J Exp Med 57:571-95.
Lancefield, R. C., and Dole, V. P. 1946. The properties of T antigens extracted from group A hemolytic streptococci. J Exp Med 84:449-71.
Lauth, X., von Köckritz-Blickwede, M., McNamara, C. W., Myskowski, S., Zinkernagel, A. S., Beall, B., Ghosh, P., Gallo, R. L., and Nizet, V. 2009. M1 protein allows group A streptococcal survival in phagocyte extracellular traps through cathelicidin inhibition. J Innate Immun 1:202-14.
Le Breton, Y., Mistry, P., Valdes, K. M., Quigley, J., Kumar, N., Tettelin, H., and McIver, K. S. 2013. Genome-wide identification of genes required for fitness of group A streptococcus in human blood. Infect Immun 81:862-75.
Lee, J. W., and Helmann, J. D. 2006. The PerR transcription factor senses H2O2 by metal-catalysed histidine oxidation. Nature 440:363-7.
Luk, E. Y., Lo, J. Y., Li, A. Z., Lau, M. C., Cheung, T. K., Wong, A. Y., Wong, M. M., Wong, C. W., Chuang, S. K., and Tsang, T. 2012. Scarlet fever epidemic, Hong Kong, 2011. Emerg Infect Dis 18:1658-61.
Makthal, N., Rastegari, S., Sanson, M., Ma, Z., Olsen, R. J., Helmann, J. D., Musser, J. M., and Kumaraswami, M. 2013. Crystal structure of peroxide stress regulator from Streptococcus pyogenes provides functional insights into the mechanism of oxidative stress sensing. J Biol Chem 288:18311-24.
Martinelli, S., Urosevic, M., Daryadel, A., Oberholzer, P. A., Baumann, C., Fey, M. F., Dummer, R., Simon, H.-U., and Yousefi, S. 2004. Induction of genes mediating interferon-dependent extracellular trap formation during neutrophil differentiation. J Biol Chem 279:44123-32.
Pancholi, V., and Fischetti, V. A. 1992. A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding-activity. J Exp Med 176:415-26.
Pancholi, V., and Fischetti, V. A. 1998. α-Enolase, a novel strong plasmin (ogen) binding protein on the surface of pathogenic streptococci. J Biol Chem 273:14503-15.
Parker, H., Albrett, A. M., Kettle, A. J., and Winterbourn, C. C. 2012. Myeloperoxidase associated with neutrophil extracellular traps is active and mediates bacterial killing in the presence of hydrogen peroxide. J Leukoc Biol 91:369-76.
Ricci, S., Janulczyk, R., and Bjorck, L. 2002. The regulator PerR is involved in oxidative stress response and iron homeostasis and is necessary for full virulence of Streptococcus pyogenes. Infect Immun 70:4968-76.
Sanderson-Smith, M. L., Dinkla, K., Cole, J. N., Cork, A. J., Maamary, P. G., McArthur, J. D., Chhatwal, G. S., and Walker, M. J. 2008. M protein-mediated plasminogen binding is essential for the virulence of an invasive Streptococcus pyogenes isolate. FASEB J 22:2715-22.
Schlievert, P. M. 1993. Role of superantigens in human disease. J Infect Dis 167:997-1002.
Staali, L., Bauer, S., Morgelin, M., Bjorck, L., and Tapper, H. 2006. Streptococcus pyogenes bacteria modulate membrane traffic in human neutrophils and selectively inhibit azurophilic granule fusion with phagosomes. Cell Microbiol 8:690-703.
Steer, A. C., Lamagni, T., Curtis, N., and Carapetis, J. R. 2012. Invasive group A streptococcal disease. Drugs 72:1213-27.
Steer, A. C., Law, I., Matatolu, L., Beall, B. W., and Carapetis, J. R. 2009. Global emm type distribution of group A streptococci: systematic review and implications for vaccine development. Lancet Infect Dis 9:611-6.
Sumby, P., Barbian, K. D., Gardner, D. J., Whitney, A. R., Welty, D. M., Long, R. D., Bailey, J. R., Parnell, M. J., Hoe, N. P., Adams, G. G., Deleo, F. R., and Musser, J. M. 2005. Extracellular deoxyribonuclease made by group A streptococcus assists pathogenesis by enhancing evasion of the innate immune response. Proc Natl Acad Sci U S A 102:1679-84.
Toukoki, C., and Gryllos, I. 2013. PolA1, a putative DNA polymerase I, is coexpressed with PerR and contributes to peroxide stress defenses of group A streptococcus. J Bacteriol 195:717-25.
Tsou, C. C., Chiang-Ni, C., Lin, Y. S., Chuang, W. J., Lin, M. T., Liu, C. C., and Wu, J. J. 2008. An iron-binding protein, Dpr, decreases hydrogen peroxide stress and protects Streptococcus pyogenes against multiple stresses. Infect Immun 76:4038-45.
Tsou, C. C., Chiang-Ni, C., Lin, Y. S., Chuang, W. J., Lin, M. T., Liu, C. C., and Wu, J. J. 2010. Oxidative stress and metal ions regulate a ferritin-like gene, dpr, in Streptococcus pyogenes. Int J Med Microbiol 300:259-64.
Uchiyama, S., Andreoni, F., Schuepbach, R. A., Nizet, V., and Zinkernagel, A. S. 2012. DNase Sda1 allows invasive M1T1 group A streptococcus to prevent TLR9-dependent recognition. PLoS Pathog 8:e1002736.
von Kockritz-Blickwede, M., Goldmann, O., Thulin, P., Heinemann, K., Norrby-Teglund, A., Rohde, M., and Medina, E. 2008. Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation. Blood 111:3070-80.
Walker, M. J., Hollands, A., Sanderson-Smith, M. L., Cole, J. N., Kirk, J. K., Henningham, A., McArthur, J. D., Dinkla, K., Aziz, R. K., Kansal, R. G., Simpson, A. J., Buchanan, J. T., Chhatwal, G. S., Kotb, M., and Nizet, V. 2007. DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection. Nat Med 13:981-5.
Wang, H., Lottenberg, R., and Boyle, M. D. 1995. Analysis of the interaction of group A streptococci with fibrinogen, streptokinase and plasminogen. Microb Pathog 18:153-66.
Weeks, C. R., and Ferretti, J. J. 1984. The gene for type A streptococcal exotoxin (erythrogenic toxin) is located in bacteriophage T12. Infect Immun 46:531-6.
Wen, Y. T., Tsou, C. C., Kuo, H. T., Wang, J. S., Wu, J. J., and Liao, P. C. 2011. Differential secretomics of Streptococcus pyogenes reveals a novel peroxide regulator (PerR)-regulated extracellular virulence factor mitogen factor 3 (MF3). Mol Cell Proteomics 10:M110 007013.
Yousefi, S., Gold, J. A., Andina, N., Lee, J. J., Kelly, A. M., Kozlowski, E., Schmid, I., Straumann, A., Reichenbach, J., Gleich, G. J., and Simon, H. U. 2008. Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense. Nat Med 14:949-53.
Zheng, P. X., Chung, K. T., Chiang-Ni, C., Wang, S. Y., Tsai, P. J., Chuang, W. J., Lin, Y. S., Liu, C. C., and Wu, J. J. 2013. Complete genome sequence of emm1 Streptococcus pyogenes A20, a strain with an intact two-component system, CovRS, isolated from a patient with necrotizing fasciitis. Genome Announc 1:e00149-12.