化膿性鏈球菌是一種重要的人類病原體，會導致多種的疾病，並會分泌許多蛋白質去影響宿主和病原體交互作用。化膿性鏈球菌感染的過程中，會導致傷口環境的酸化，形成膿瘡或壞死性病變，但是目前很少研究提到化膿性鏈球菌在酸性環境下如何調控毒性蛋白質的表現，增加細菌的入侵能力。因此，本研究的目的是要找出受酸性影響的化膿性鏈球菌分泌性蛋白質。首先，我們收集在中性條件(pH7.5)和微酸性條件(pH6.0)培養的化膿性鏈球菌分泌蛋白質樣品。進一步量測其生長曲線，發現化膿性鏈球菌在中性和酸性環境下的生長曲線並無太大差異。但經由一維膠體電泳分析，可以明顯看出中性和酸性環境下蛋白質有表現量上的差異。利用堆疊膠體電泳(stacking gel)將中性和酸性環境下取得的蛋白質回收，再以非標記串聯式質譜分析方法得到中性和酸性環境下的蛋白質體資訊。在本研究當中，我們總共鑑定到172個蛋白質受到環境酸化的影響，在連續兩個時間點以上的表現量有相同的顯著性差異，其中篩選出45 (26%)個蛋白質被預測可能是分泌蛋白質。根據過去研究的分析比對，分別對應到21個蛋白質與免疫抗性、營養源的攝取、對宿主的黏附力、導致凝血功能障礙及組織損傷等致病機制相關，其中已知受酸化刺激會誘導表現的蛋白質：化膿性鏈球菌熱源性外毒素B和線毛相關蛋白也在本研究中被觀察到。本研究鑑定到許多受酸化影響的化膿性鏈球菌致病相關分泌性蛋白質，此非標記蛋白質體的分析數據對於瞭解化膿性鏈球菌感染時引起的環境酸化的相關機轉是很重要的。

Streptococcus pyogenes is an important human pathogen that causes a broad range of diseases. S. pyogenes secretes a number of proteins involved in host-pathogen interactions. The acidic environment in the infected site can trigger S. pyogenes to generate an abscess or a necrotic lesion. But, little is known about how environmental acidification regulates expression of many virulence factors in S. pyogenes for severe infections. Therefore, the objective of this study was to identify the acid-influenced secreted proteins of S. pyogenes. First, the secretome samples of S. pyogenes were collected from neutral culture conditions (pH7.5) and from acidic culture condition (pH6.0) at designed sampling time points. The optical density was measured for establishment of growth curve. The growth curves were similar between neutral and acidic culture conditions. The protein patterns on SDS-PAGE show dissimilarity between the secretomes from acidic and neutral conditions. The secretome samples were cleaned up by a stacking gel and were analyzed by label-free LC-MS/MS analysis. The dynamic label-free LC-MS/MS profiling identified 172 proteins which are influenced by environmental acidification at least two consecutive time points. Among these, 45 (26%) the identified proteins are predicted secreted proteins. According to pathogenesis evidences, 21 proteins are pathogenesis-related protein and can influence immune evasion, nutrient acquisition, cell adhesion, hemolysis, and tissue damage. The known acid-induced proteins, SpeB and pilus-related protein are also observed in this study. In this study, we identified many acid-influenced secreted proteins related to Streptococcus pyogenes pathogenesis. Our label-free proteomic data is crucial for realizing mechanisms related to environmental acidification of group A streptococcal infection.

Allhorn, M., Briceno, J. B., Baudino, L., Lood, C., Olsson, M. L., Izui, S., and Collin, M. (2010). "The IgG-specific endoglycosidase EndoS inhibits both cellular and complement-mediated autoimmune hemolysis." Blood 115(24): 5080-5088.

Allhorn, M., Olsen, A. and Collin, M. (2008). "EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity." BMC Microbiol 8: 3.

Anderson, N. L. and Anderson, N. G. (1998). "Proteome and proteomics: new technologies, new concepts, and new words." Electrophoresis 19(11): 1853-61.

Becherelli, M., Manetti, A. G., Buccato, S., Viciani, E., Ciucchi, L., Mollica, G., Grandi, G. and Margarit, I. (2012). "The ancillary protein 1 of Streptococcus pyogenes FCT-1 pili mediates cell adhesion and biofilm formation through heterophilic as well as homophilic interactions." Mol Microbiol 83(5): 1035-1047.

Bisno, A. L., Brito, M. O. and Collins, C. M. (2003). "Molecular basis of group A streptococcal virulence." Lancet Infect Dis 3(4): 191-200.

Biswas, I., Drake, L. and Biswas, S. (2007). "Regulation of gbpC expression in Streptococcus mutans." J Bacteriol 189(18): 6521-6531.

Boekhorst, J., Wels, M., Kleerebezem, M. and Siezen, R. J. (2006). "The predicted secretome of Lactobacillus plantarum WCFS1 sheds light on interactions with its environment." Microbiology 152(Pt 11): 3175-3183.

Bugrysheva, J., Froehlich, B. J., Freiberg, J. A. and Scott, J. R. (2011). "Serine/threonine protein kinase Stk is required for virulence, stress response, and penicillin tolerance in Streptococcus pyogenes." Infect Immun 79(10): 4201-4209.

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(11): 685-694.

Chaussee, M. S., Sylva, G. L., Sturdevant, D. E., Smoot, L. M., Graham, M. R., Watson, R. O. and Musser, J. M. (2002). "Rgg influences the expression of multiple regulatory loci to coregulate virulence factor expression in Streptococcus pyogenes." Infect Immun 70(2): 762-770.

Chiu, K. H., Chang, Y. H., Wu, Y. S., Lee, S. H. and Liao, P. C. (2011). "Quantitative secretome analysis reveals that COL6A1 is a metastasis-associated protein using stacking gel-aided purification combined with iTRAQ labeling." J Proteome Res 10(3): 1110-1125.

Chuan, N. C., Zheng, P. X., Tsai, P. J., Chuang, W. J., Lin, Y. S., Liu, C. C. and Wu, J. J. (2012). "Environmental pH changes, but not the LuxS signalling pathway, regulate SpeB expression in M1 group A streptococci." J Med Microbiol 61(1): 16-22.

Collin, M. and Olsén, A. (2001). "EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG." EMBO J 20(12): 3046-3055.

Cox, J. and Mann, M. (2008). "MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification." Nat Biotechnol 26(12): 1367-1372.

Cunningham, M. W. (2000). "Pathogenesis of Group A Streptococcal Infections." Clin Microbiol Rev 13(3): 470-511.

Dalton, T. L. and Scott, J. R. (2004). "CovS inactivates CovR and is required for growth under conditions of general stress in Streptococcus pyogenes." J Bacteriol 186(12): 3928-3937.

Daniel, H. (2002). "Genomics and proteomics: importance for the future of nutrition research." Br J Nutr 87(Suppl 2): S305-11.

Federle, M. (2012). "Pathogenic streptococci speak, but what are they saying?" Virulence 3(1): 92-94.

Gao, B. B., Stuart, L. and Feener, E. P. (2008). "Label-free quantitative analysis of one-dimensional PAGE LC/MS/MS proteome: application on angiotensin II-stimulated smooth muscle cells secretome." Mol Cell Proteomics 7(12): 2399-409.

Gase, K., Ferretti, J. J., Primeaux, C. and McShan, M. W. (1999). "Identification, cloning, and expression of the CAMP factor gene (cfa) of group A streptococci." Infect Immun 67(9): 4725-4731.

Hoe, N. P., Ireland, R. M., DeLeo, F. R., Gowen, B. B., Dorward, D. W., Voyich, J. M., Liu, M., Burns, E. H. Jr., Culnan, D. M., Bretscher, A. and Musser, J. M. (2002). "Insight into the molecular basis of pathogen abundance: group A Streptococcus inhibitor of complement inhibits bacterial adherence and internalization into human cells." Proc Natl Acad Sci U S A 99(11): 7646-7651.

Hynes, W., Johnson, C. and Stokes, M. (2009). "A single nucleotide mutation results in loss of enzymatic activity in the hyaluronate lyase gene of Streptococcus pyogenes." Microb Pathog 47(6): 308-313.

Hytonen, J., Haataja, S. and Finne, J. (2003). "Streptococcus pyogenes glycoprotein-binding strepadhesin activity is mediated by a surface-associated carbohydrate-degrading enzyme, pullulanase." Infect Immun 71(2): 784-793.

Kimura, K. R., Nakata, M., Sumitomo, T., Kreikemeyer, B., Podbielski, A., Terao, Y. and Kawabata, S. (2012). "Involvement of T6 pili in biofilm formation by serotype M6 Streptococcus pyogenes." J Bacteriol 194(4): 804-812.

Kunitomo, E., Terao, Y., Okamoto, S., Rikimaru, T., Hamada, S. and Kawabata, S. (2008). "Molecular and biological characterization of histidine triad protein in group A streptococci." Microbes Infect 10(4): 414-423.

Lange, V., Malmström, J. A., Didion, J., King, N. L., Johansson, B. P., Schäfer, J., Rameseder, J., Wong, C. H., Deutsch, E. W., Brusniak, M. Y., Bühlmann, P., Björck, L., Domon, B. and Aebersold, R. (2008). "Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoring." Mol Cell Proteomics 7: 1489-1500.

Linke, C., Siemens, N., Oehmcke, S., Radjainia, M., Law, R. H., Whisstock, J. C., Baker, E. N. and Kreikemeyer, B. (2012). "The extracellular protein factor Epf from Streptococcus pyogenes is a cell surface adhesin that binds to cells through an N-terminal domain containing a carbohydrate-binding module." J Biol Chem 287(45): 38178-38189.

Loughman, J. A. and Caparon, M. (2006). "Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: a model for in vivo gene expression." J Bacteriol 188(2): 399-408.

Ma, Y., Bryant, A. E., Salmi, D. B., Hayes-Schroer, S. M., McIndoo, E., Aldape, M. J. and Stevens, D. L. (2006). "Identification and characterization of bicistronic speB and prsA gene expression in the group A Streptococcus." J Bacteriol 188(21): 7626-7634.

Manetti, A. G., Köller, T., Becherelli, M., Buccato, S., Kreikemeyer, B., Podbielski, A., Grandi, G. and Margarit, I. (2010). "Environmental acidification drives S. pyogenes pilus expression and microcolony formation on epithelial cells in a FCT-dependent manner." PLoS One 5(11): e13864.

Neely, M. N., Lyon, W. R., Runft, D. L. and Caparon, M. (2003). "Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes." J Bacteriol 185(17): 5166-5174.

Nyberg, P., Sakai, T., Cho, K. H., Caparon, M. G., Fässler, R. and Björck, L. (2004). "Interactions with fibronectin attenuate the virulence of Streptococcus pyogenes." EMBO J 23(10): 2166-2174.

Ohara-Nemoto, Y., Sasaki, M., Kaneko, M., Nemoto, T. and Ota, M. (1994). "Cysteine protease activity of streptococcal pyrogenic exotoxin B." Can J Microbiol 40(11): 930-936.

Olsen, R. J. and Musser, J. M. (2010). "Molecular pathogenesis of necrotizing fasciitis." Annu Rev Pathol 5: 1-31.

Olsen, R. J., Shelburne, S. A. and Musser, J. M. (2009). "Molecular mechanisms underlying group A streptococcal pathogenesis." Cell Microbiol 11(1): 1-12.

Port, G. C. and Freitag, N. E. (2007). "Identification of novel Listeria monocytogenes secreted virulence factors following mutational activation of the central virulence regulator, PrfA." Infect Immun 75(12): 5886-5897.

Salim, K. Y., de Azavedo, J. C., Bast, D. J. and Cvitkovitch, D. G. (2007). "Role for sagA and siaA in quorum sensing and iron regulation in Streptococcus pyogenes." Infect Immun 75(10): 5011-5017.

Shelburne, S. A. 3rd., Fang, H., Okorafor, N., Sumby, P., Sitkiewicz, I., Keith, D., Patel, P., Austin, C., Graviss, E. A., Musser, J. M. and Chow, D. C. (2007). "MalE of group A Streptococcus participates in the rapid transport of maltotriose and longer maltodextrins." J Bacteriol 189(7): 2610-2617.

Shelburne, S. A. 3rd., Keith, D. B., Davenport, M. T., Beres, S. B., Carroll, R. K. and Musser, J. M. (2009). "Contribution of AmyA, an extracellular alpha-glucan degrading enzyme, to group A streptococcal host-pathogen interaction." Mol Microbiol 74(1): 159-174.

Simmen, H. P., Battaglia, H., Giovanoli, P. and Blaser, J. (1994). " Analysis of pH, pO2 and pCO2 in drainage fluid allows for rapid detection of infectious complications during the follow-up period after abdominal surgery." Infection 22(6): 14-17.

Starr, C. R. and Engleberg, N. C. (2006). "Role of hyaluronidase in subcutaneous spread and growth of group A streptococcus." Infect Immun 74(1): 40-48.

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(5): 1679-1684.

Tsai, P. J., Chen, Y. H., Hsueh, C. H., Hsieh, H. C., Liu, Y. H., Wu, J. J. and Tsou C. C. (2006). "Streptococcus pyogenes induces epithelial inflammatory responses through NF-kappaB/MAPK signaling pathways." Microbes Infect 8(6): 1440-1449.

von Pawel-Rammingen, U. and Bjorck, L. (2003). "IdeS and SpeB: immunoglobulin-degrading cysteine proteinases of Streptococcus pyogenes." Curr Opin Microbiol 6(1): 50-55.

Wu, W. W. Wang, G., Baek, S. J. and Shen, R. F. (2006). "Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF." J Proteome Res 5(3): 651-8.

Zhang, M. J., Zhao, F., Xiao, D., Gu, Y. X., Meng, F. L., He, L. H. and Zhang, J. Z. (2009). "Comparative proteomic analysis of passaged Helicobacter pylori." J Basic Microbiol 49(5): 482-490.