化膿性鏈球菌(Streptococcus pyogenes)，又稱為A群鏈球菌(group A streptococcus, GAS)，為革蘭氏陽性、會產生β型溶血的人類致病菌，臨床上會造成許多疾病，輕微症狀者為咽喉炎或咽頰炎，但也會造成嚴重疾病例如壞死性筋膜炎、鏈球性毒性休克症候群。過去的研究統計指出每年約有3400萬的人受到GAS的感染，且至少造成5萬人死亡，此外，從1980年起，侵襲性的GAS感染在全球各地開始大幅增加，雖然目前臨床上的抗生素使用可以有效治療GAS造成的疾病，但是由於突變珠以及侵襲性菌株的出現，使得GAS感染在臨床上仍然可以逃脫抗生素的攻擊，進而造成嚴重的侵襲性致命疾病。在過去的研究中GAS都被認為屬於細胞外感染的致病菌，但是近來的研究發現GAS可以在細胞內存活並且複製，進而造成嚴重的侵襲性疾病，已知的研究中顯示，GAS可以在巨噬細胞以及內皮細胞(endothelial cell)中利用某些機制逃避免疫系統的攻擊，而在細胞內存活，也有越來越多研究顯示NAD-glycohydrolase (NADase)對於GAS在細胞內的生存扮演一個重要的角色，然而，NADase在分子機轉層面的調控尚未清楚，而在最近的研究顯示，NADase的表現是受到轉錄因子Rgg的調控，從過去的研究可以知道Rgg會調控多種GAS內的毒性基因表現，其中包括了slo、speB以及nga。在本篇研究中，主要探討Rgg調控NADase的分子機制，以及Rgg與nga promoter之間的交互作用。我們希望以結構為基礎，進一步去探討Rgg與DNA之間的交互作用。除此之外，我們也探討了CovRS與Rgg之間的關係，在過去的研究中顯示，GAS中的two-component system CovRS調控了約15%的基因表現，其中包括了毒性基因以及環境適應相關的基因表現，因此可以知道CovRS對於GAS在進行感染以及適應環境是扮演一個重要的腳色。總結來說，我們利用結構解析的方式去了解Rgg在GAS中的分子機轉，除此之外，也探討了CovRS、Rgg以及NADase之間相互調控的關係，最後，我們希望以結構為基礎，在未來能夠設計針對參與在分子機轉調控中相關的蛋白抑制劑，透過調節GAS中的致病相關基因，降低GAS中毒性因子的表現，達到有效降低GAS在人體上造成嚴重疾病以及治療的效果。

Group A streptococcus (GAS) is a beta-hemolytic Gram-positive coccus which is renowned for its highly aggressiveness to destruct host tissues. GAS causes various mild human diseases such as pharyngitis and impetigo, while invasive GAS infections in deep tissues cause severe life-threatening diseases, such as necrotizing fasciitis and streptococcal toxic shock syndrome. According to the reports, there are at least 34 million people infected by GAS-related disease, and the infection causes more than 50 thousand deaths per year. Since 1980s, there was a global increase in invasive GAS infection, and invasive GAS infections cause severe life-threatening diseases. In spite of the antibiotics treatment, the life-threatening diseases still exist due to the mutation and invasive strains. Although GAS was considered to be the extracellular pathogens, recent studies found GAS can survive intracellularly that might be crucial for development of invasive diseases. Previous studies found that invasive strains secrete NAD-glycohydrolase (NADase or nga) which is essential for GAS survival in endothelial cell. However, how NADase is regulated at molecular level has not yet been elucidated. Recent studies also determined that the expression of nga might be regulated by the transcriptional regulator Rgg which has been reported to affect the transcription of various virulence factors, including slo, speB and nga. In this study, we aim to understand the mechanisms of how nga is regulated at molecular level and how Rgg acts on the promoter of nga promoter. Our goal is to study the structure-function relationship of Rgg by determining the structure of Rgg and understanding the molecular interactions between Rgg and DNA. In addition, we have also revealed the molecular regulation of Rgg by CovRS, a two-component system CovRS regulated about 15% of gene expression in GAS and was found to play a crucial role in GAS pathogenesis. In summary, my thesis work has delineated the molecular regulatory network between CovRS, Rgg and NADase to understand the regulation pathway and provide insights into future therapeutic design aimed at modulating the activity of regulators for inhibiting virulence factors expression to treat severely invasive GAS infections.

1. Walker, M. J., T. C. Barnett, J. D. McArthur, J. N. Cole, C. M. Gillen, A. Henningham, K. S. Sriprakash, M. L. Sanderson-Smith and V. Nizet. Disease manifestations and pathogenic mechanisms of group A streptococcus. Clinical Microbiology Reviews 27, 264-301 (2014).
2. Brouwer, S., Barnett, T.C., Rivera-Hernandez, T., Rohde, M. & Walker, M.J. Streptococcus pyogenes adhesion and colonization. FEBS letters 590, 3739-3757 (2016).
3. Excler, J.L. & Kim, J.H. Accelerating the development of a group A streptococcus vaccine: an urgent public health need. Clin Exp Vaccine Res 5, 101-107 (2016).
4. DALYs, G. B. D., H. Collaborators, C. J. Murray, R. M. Barber, K. J. Foreman, A. Abbasoglu Ozgoren, F. Abd-Allah, S. F. Abera, V. Aboyans, J. P. Abraham, I. Abubakar, L. J. Abu-Raddad, N. M. Abu-Rmeileh, et al. Global, regional, and national disability-adjusted life years (DALYs) for 306 diseases and injuries and healthy life expectancy (HALE) for 188 countries, 1990-2013: quantifying the epidemiological transition. Lancet 386, 2145-2191 (2015).
5. Lennon, D. Acute rheumatic fever in children: recognition and treatment. Paediatr Drugs 6, 363-373 (2004).
6. Barnett, T. C., J. N. Cole, T. Rivera-Hernandez, A. Henningham, J. C. Paton, V. Nizet and M. J. Walker. Streptococcal toxins: role in pathogenesis and disease. Cellular Microbiology 17, 1721-1741 (2015).
7. Bastiat-Sempe, B., Love, J.F., Lomayesva, N. & Wessels, M.R. Streptolysin O and NAD-glycohydrolase prevent phagolysosome acidification and promote group A streptococcus survival in macrophages. mBio 5, e01690-01614 (2014).
8. Lu, S. L., C. F. Kuo, H. W. Chen, Y. S. Yang, C. C. Liu, R. Anderson, J. J. Wu and Y. S. Lin. Insufficient acidification of autophagosomes facilitates group A streptococcus survival and growth in endothelial cells. mBio 6, e01435-01415 (2015).
9. Bricker, A.L., Carey, V.J. & Wessels, M.R. Role of NADase in virulence in experimental invasive group A streptococcal infection. Infection and Immunity 73, 6562-6566 (2005).
10. O'Seaghdha, M. & Wessels, M.R. Streptolysin O and its co-toxin NAD-glycohydrolase protect group A streptococcus from xenophagic killing. PLoS Pathogens 9, e1003394 (2013).
11. Sharma, O., O'Seaghdha, M., Velarde, J.J. & Wessels, M.R. NAD+-glycohydrolase promotes intracellular survival of group A streptococcus. PLoS Pathogens 12, e1005468 (2016).
12. Stevens, D.L., Salmi, D.B., McIndoo, E.R. & Bryant, A.E. Molecular epidemiology of nga and NAD glycohydrolase/ADP-ribosyltransferase activity among Streptococcus pyogenes causing streptococcal toxic shock syndrome. The Journal of Infectious Diseases 182, 1117-1128 (2000).
13. Sumby, P., S. F. Porcella, A. G. Madrigal, K. D. Barbian, K. Virtaneva, S. M. Ricklefs, D. E. Sturdevant, M. R. Graham, J. Vuopio-Varkila, N. P. Hoe and J. M. Musser. Evolutionary origin and emergence of a highly successful clone of serotype M1 group a streptococcus involved multiple horizontal gene transfer events. The Journal of Infectious Diseases 192, 771-782 (2005).
14. Tatsuno, I., J. Sawai, A. Okamoto, M. Matsumoto, M. Minami, M. Isaka, M. Ohta and T. Hasegawa. Characterization of the NAD-glycohydrolase in streptococcal strains. Microbiology 153, 4253-4260 (2007).
15. Sulavik, M.C. & Clewell, D.B. Rgg is a positive transcriptional regulator of the Streptococcus gordonii gtfG gene. Journal of Bacteriology 178, 5826-5830 (1996).
16. Dmitriev, A. V., E. J. McDowell, K. V. Kappeler, M. A. Chaussee, L. D. Rieck and M. S. Chaussee. The Rgg regulator of Streptococcus pyogenes influences utilization of nonglucose carbohydrates, prophage induction, and expression of the NAD-glycohydrolase virulence operon. Journal of Bacteriology 188, 7230-7241 (2006).
17. Zheng, F., H. Ji, M. Cao, C. Wang, Y. Feng, M. Li, X. Pan, J. Wang, Y. Qin, F. Hu and J. Tang. Contribution of the Rgg transcription regulator to metabolism and virulence of Streptococcus suis serotype 2. Infection and Immunity 79, 1319-1328 (2011).
18. Loughman, J.A. & Caparon, M.G. Contribution of invariant residues to the function of Rgg family transcription regulators. Journal of Bacteriology 189, 650-655 (2007).
19. Neely, M.N., Lyon, W.R., Runft, D.L. & Caparon, M. Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes. Journal of Bacteriology 185, 5166-5174 (2003).
20. Loughman, J.A. & Caparon, M.G. A novel adaptation of aldolase regulates virulence in Streptococcus pyogenes. The EMBO journal 25, 5414-5422 (2006).
21. Anbalagan, S., Dmitriev, A., McShan, W.M., Dunman, P.M. & Chaussee, M.S. Growth phase-dependent modulation of Rgg binding specificity in Streptococcus pyogenes. Journal of Bacteriology 194, 3961-3971 (2012).
22. Cole, J.N., Barnett, T.C., Nizet, V. & Walker, M.J. Molecular insight into invasive group A streptococcal disease. Nature Reviews. Microbiology 9, 724-736 (2011).
23. Lee, S.F., Delaney, G.D. & Elkhateeb, M. A two-component covRS regulatory system regulates expression of fructosyltransferase and a novel extracellular carbohydrate in Streptococcus mutans. Infection and Immunity 72, 3968-3973 (2004).
24. Friaes, A., Pato, C., Melo-Cristino, J. & Ramirez, M. Consequences of the variability of the CovRS and RopB regulators among Streptococcus pyogenes causing human infections. Sci Rep 5, 12057 (2015).
25. Tatsuno, I., Okada, R., Zhang, Y., Isaka, M. & Hasegawa, T. Partial loss of CovS function in Streptococcus pyogenes causes severe invasive disease. BMC Res Notes 6, 126 (2013).
26. Stephenson, K. & Hoch, J.A. Virulence- and antibiotic resistance-associated two-component signal transduction systems of Gram-positive pathogenic bacteria as targets for antimicrobial therapy. Pharmacology & Therapeutics 93, 293-305 (2002).
27. Federle, M.J., McIver, K.S. & Scott, J.R. A response regulator that represses transcription of several virulence operons in the group A streptococcus. Journal of Bacteriology 181, 3649-3657 (1999).
28. Chiang-Ni, C., Chu, T.P., Wu, J.J. & Chiu, C.H. Repression of Rgg but not upregulation of LacD.1 in emm1-type covS mutant mediates the SpeB repression in group A streptococcus. Frontiers in Microbiology 7, 1935 (2016).
29. Fleuchot, B., A. Guillot, C. Mezange, C. Besset, E. Chambellon, V. Monnet and R. Gardan. Rgg-associated SHP signaling peptides mediate cross-talk in streptococci. PloS One 8, e66042 (2013).
30. Rocha-Estrada, J., Aceves-Diez, A.E., Guarneros, G. & de la Torre, M. The RNPP family of quorum-sensing proteins in Gram-positive bacteria. Applied Microbiology and Biotechnology 87, 913-923 (2010).
31. Parashar, V., Aggarwal, C., Federle, M.J. & Neiditch, M.B. Rgg protein structure-function and inhibition by cyclic peptide compounds. Proceedings of the National Academy of Sciences of the United States of America 112, 5177-5182 (2015).
32. Colledge, V. L., M. J. Fogg, V. M. Levdikov, A. Leech, E. J. Dodson and A. J. Wilkinson. Structure and organisation of SinR, the master regulator of biofilm formation in Bacillus subtilis. Journal of Molecular Biology 411, 597-613 (2011).
33. Grenha, R., L. Slamti, M. Nicaise, Y. Refes, D. Lereclus and S. Nessler. Structural basis for the activation mechanism of the PlcR virulence regulator by the quorum-sensing signal peptide PapR. Proceedings of the National Academy of Sciences of the United States of America 110, 1047-1052 (2013).
34. Makthal, N., M. Gavagan, H. Do, R. J. Olsen, J. M. Musser and M. Kumaraswami. Structural and functional analysis of RopB: a major virulence regulator in Streptococcus pyogenes. Molecular Microbiology 99, 1119-1133 (2016).
35. Mertens, H.D. & Svergun, D.I. Structural characterization of proteins and complexes using small-angle X-ray solution scattering. J Struct Biol 172, 128-141 (2010).
36. Putnam, C.D., Hammel, M., Hura, G.L. & Tainer, J.A. X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q Rev Biophys 40, 191-285 (2007).
37. Rambo, R.P. & Tainer, J.A. Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers 95, 559-571 (2011).
38. Rambo, R.P. & Tainer, J.A. Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small-angle X-ray scattering. Curr Opin Struct Biol 20, 128-137 (2010).
39. Chaussee, M.S., Somerville, G.A., Reitzer, L. & Musser, J.M. Rgg coordinates virulence factor synthesis and metabolism in Streptococcus pyogenes. Journal of Bacteriology 185, 6016-6024 (2003).
40. Schneidman-Duhovny, D., Hammel, M., Tainer, J.A. & Sali, A. Accurate SAXS profile computation and its assessment by contrast variation experiments. Biophys J 105, 962-974 (2013).
41. Schneidman-Duhovny, D., Hammel, M., Tainer, J.A. & Sali, A. FoXS, FoXSDock and MultiFoXS: Single-state and multi-state structural modeling of proteins and their complexes based on SAXS profiles. Nucleic acids research 44, W424-429 (2016).
42. Graham, M. R., L. M. Smoot, C. A. Migliaccio, K. Virtaneva, D. E. Sturdevant, S. F. Porcella, M. J. Federle, G. J. Adams, J. R. Scott and J. M. Musser. Virulence control in group A streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling. Proceedings of the National Academy of Sciences of the United States of America 99, 13855-13860 (2002).
43. Lin, C. S., S. Y. Chao, M. Hammel, J. C. Nix, H. L. Tseng, C. C. Tsou, C. H. Fei, H. S. Chiou, U. S. Jeng, Y. S. Lin, W. J. Chuang, J. J. Wu and S. Wang. Distinct structural features of the peroxide response regulator from group A streptococcus drive DNA binding. PloS One 9, e89027 (2014).