在革蘭氏陽性細菌中，與毒力因子相關的表面蛋白質(surface protein)主要藉由半胱氨酸蛋白酶(cysteine protease)類的分選酶(sortase)將此蛋白質送至細胞壁上，其在細菌感染時扮演重要角色。而困難梭狀桿菌Clostridium difficile (C. difficile)與其他眾多陽性菌不同，僅具有一種分選sortase B (SrtB)。C. difficile SrtB主要藉由辨認序列上含有(S/P)PXTG序列的受質蛋白質(substrate protein)並附著至細胞壁上。C. difficile可以導致嚴重的腸道疾病，例如腹瀉，發炎性腸道疾病(IBD)和偽膜性結腸炎，這些症狀主要因腸道微生物群落在抗生素治療下破壞所導致的。因此，開發能替代抗生素的藥物是必要的。Sortase被認為是可替代性藥物的標的目標，因為它不影響細菌生長，若發展出其有效抑制劑後，既可調節C. difficile的細胞表面蛋白質，並不對環境中其他細菌施加選擇性壓力進而導致抗藥性的發生。在革蘭氏陽性菌中，SrtB具有高度的結構相似性，但在受質(substrate)上卻辨認不同的序列。在近期研究中，我們實驗室已經成功的解出SrtB蛋白的結晶結構。然而，目前尚未清楚SrtB是如何辨別相關表面蛋白質的特定序列並將其附著於C. difficile的細胞壁上。因此，本篇研究主要透過定位突變(site-directed mutagenesis)和螢光共振能量轉移測定(fluorescence resonance energy transfer) (FRET-based assay)以了解SrtB對於substrate辨認專一性, 分子作用以及參與在SrtB活性位點中的重要氨基酸。在實驗結果中，C. difficile的SrtB會形成Cys209-Arg217-His116的三位點(Triad)以攻擊受質上分選信號(sorting signal)肽鍵。而在實驗結果及晶體研究中發現，Cys209和Arg217在調控酵素活性中為重要的氨基酸殘基(residue)。Cys209為主要提供硫醇基團(thiol group)以得到電子攻擊sorting signal的肽鍵。Arg217主要穩定受質結合以形成oxyanion hole並促進催化機制的完成。此外，在活性位點中Cys周圍的Ser第一次被發現能參與在C. difficile以及金黃色葡萄球菌Staphylococcus aureus中SrtB的催化機制。並且在序列比對下，其他陽性菌中的SrtB在相似位點上也發現此一高度保留性的Ser殘基。此外，針對已知S. aureus的SrtB活性位點合成並篩選數種小分子抑製劑。以FRET-based assay偵測SrtB活性並加上最低抑菌濃度測定(minimum inhibitory concentration; MIC)和最低殺菌濃度測定(minimum bactericidal concentration; MBC)，結果顯示所篩選出來有效的抑製劑中，主要是藉由抑制Cd-SrtBΔN26活性而不是透過殺死細菌來抑制其作用。總結而論，透過理解SrtB與其受質間特異性，以了解SrtB的分子作用，並確定活性位點中的殘基進而能發展出更專一以及有效的抑製劑來治療C. difficile相關疾病。

In Gram-positive bacteria, virulence-associated surface proteins to the peptidoglycan cell wall is anchored by the sortase, a cysteine transpetidase, which plays an important role during infection. In contrast to other bacteria, Clostridium difficile possesses only sortase B (SrtB) which recognized the conserved (S/P)PXTG motif of substrate proteins for the process of attachment. Hospitalized patients receiving antibiotics, which often disrupt intestinal microflora, belong to the high risk population for C. difficile infection resulting in severe intestinal diseases, such as diarrhea, inflammatory bowel disease (IBD), and pseudomembranous colitis. Thus, developing an alternative anti-infective drug other than conventional antibiotics is necessary. Sortase has been recognized as a promising therapeutic target because it is not required for bacterial growth, thus sortase inhibitors could modulate the bacterial virulence without placing selective pressure on bacteria. It is still unclear how SrtB recognizes the specific motif of substrate proteins and attaches them to the peptidoglycan in C. difficile. Hence, the aim is to understand the molecular action of SrtB. The key residue of active site in C. difficile SrtBΔN26 was identified by mutagenesis studies and fluorescence resonance energy transfer-based assay. The active site of Cd-SrtB was monitored and was found to form a triad of Cys209-Arg217-His116 which attacks the peptide of the sorting signal. The experiment demonstrated that the important residues were Cys209 and Arg217 in the active site of Cd-SrtBΔN26. Based on the crystal structural results of mutants in Cd-SrtB ΔN26 and previous studies, it has been established that Cys209 provides the charged residue with the thiol group necessary to attack the peptide bond of the sorting signal. Arg217 is also essential for stabilizing substrate-binding to form the oxyanion hole and promote completion of the catalytic mechanism. Additionally, a Ser residue located near the Cys residue at the active site was first discovered to be involved in the catalytic mechanism of SrtB in C. difficile and S.aureus. This residue of Ser is conserved in SrtB from other Gram-positive bacteria. Once the molecular mechanism is clear, the next stage of work is the design of specific SrtB inhibitors to suppress the anchoring of surface proteins associated with bacterial adhesion. Consequently, several synthesized small-molecule inhibitors were screened against SrtB activity for developing structure-based drugs. The results revealed that the selected inhibitors function mainly by inhibiting the enzymatic activity of Cd-SrtBΔN26 instead of killing the bacteria. In summary, our work has contributed to a better understanding of sortase-substrate specificity, provided the molecular actions of C. difficile SrtB to define the key residues in the active site and identified effective inhibitors.

1 Leffler, D. A. & Lamont, J. T. Clostridium difficile infection. N Engl J Med 372, 1539-1548, doi:10.1056/NEJMra1403772 (2015).
2 Rupnik, M., Wilcox, M. H. & Gerding, D. N. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 7, 526-536, doi:10.1038/nrmicro2164 (2009).
3 Napolitano, L. M. & Edmiston, C. E., Jr. Clostridium difficile disease: Diagnosis, pathogenesis, and treatment update. Surgery 162, 325-348, doi:10.1016/j.surg.2017.01.018 (2017).
4 Dubberke, E. R. et al. Strategies to prevent Clostridium difficile infections in acute care hospitals. Infect Control Hosp Epidemiol 29 Suppl 1, S81-92, doi:10.1086/591065 (2008).
5 Barra-Carrasco, J. & Paredes-Sabja, D. Clostridium difficile spores: a major threat to the hospital environment. Future Microbiol 9, 475-486, doi:10.2217/fmb.14.2 (2014).
6 Dubberke, E. R. et al. Strategies to prevent Clostridium difficile infections in acute care hospitals: 2014 Update. Infect Control Hosp Epidemiol 35, 628-645, doi:10.1086/676023 (2014).
7 Goudarzi, M., Seyedjavadi, S. S., Goudarzi, H., Mehdizadeh Aghdam, E. & Nazeri, S. Clostridium difficile Infection: Epidemiology, Pathogenesis, Risk Factors, and Therapeutic Options. Scientifica (Cairo) 2014, 916826, doi:10.1155/2014/916826 (2014).
8 Rineh, A., Kelso, M. J., Vatansever, F., Tegos, G. P. & Hamblin, M. R. Clostridium difficile infection: molecular pathogenesis and novel therapeutics. Expert Rev Anti Infect Ther 12, 131-150, doi:10.1586/14787210.2014.866515 (2014).
9 Rupnik, M. et al. Characterization of polymorphisms in the toxin A and B genes of Clostridium difficile. FEMS Microbiol Lett 148, 197-202 (1997).
10 Katyal, R., Vaishnavi, C. & Singh, K. Faecal excretion of brush border membrane enzymes in patients with Clostridium difficile diarrhoea. Indian journal of medical microbiology 20, 178-182 (2002).
11 Altaie, S. S., Meyer, P. & Dryja, D. Comparison of two commercially available enzyme immunoassays for detection of Clostridium difficile in stool specimens. J Clin Microbiol 32, 51-53 (1994).
12 Vaishnavi, C. Clinical spectrum & pathogenesis of Clostridium difficile associated diseases. The Indian journal of medical research 131, 487-499 (2010).
13 Poxton, I. R., McCoubrey, J. & Blair, G. The pathogenicity of Clostridium difficile. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 7, 421-427 (2001).
14 Barbut, F. et al. Clinical features of Clostridium difficile-associated infections and molecular characterization of strains: results of a retrospective study, 2000-2004. Infect Control Hosp Epidemiol 28, 131-139, doi:10.1086/511794 (2007).
15 Peery, A. F. et al. Burden of gastrointestinal disease in the United States: 2012 update. Gastroenterology 143, 1179-1187 e1171-1173, doi:10.1053/j.gastro.2012.08.002 (2012).
16 Asempa, T. E. & Nicolau, D. P. Clostridium difficile infection in the elderly: an update on management. Clin Interv Aging 12, 1799-1809, doi:10.2147/CIA.S149089 (2017).
17 Garey, K. W., Sethi, S., Yadav, Y. & DuPont, H. L. Meta-analysis to assess risk factors for recurrent Clostridium difficile infection. J Hosp Infect 70, 298-304, doi:10.1016/j.jhin.2008.08.012 (2008).
18 Honda, H., Yamazaki, A., Sato, Y. & Dubberke, E. R. Incidence and mortality associated with Clostridium difficile infection at a Japanese tertiary care center. Anaerobe 25, 5-10, doi:10.1016/j.anaerobe.2013.10.004 (2014).
19 Johnson, S. Recurrent Clostridium difficile infection: a review of risk factors, treatments, and outcomes. J Infect 58, 403-410, doi:10.1016/j.jinf.2009.03.010 (2009).
20 Chung, C. H. et al. Clostridium difficile infection at a medical center in southern Taiwan: incidence, clinical features and prognosis. J Microbiol Immunol Infect 43, 119-125, doi:10.1016/S1684-1182(10)60019-9 (2010).
21 Cohen, S. H. et al. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the infectious diseases society of America (IDSA). Infect Control Hosp Epidemiol 31, 431-455, doi:10.1086/651706 (2010).
22 Debast, S. B., Bauer, M. P., Kuijper, E. J., European Society of Clinical, M. & Infectious, D. European Society of Clinical Microbiology and Infectious Diseases: update of the treatment guidance document for Clostridium difficile infection. Clin Microbiol Infect 20 Suppl 2, 1-26, doi:10.1111/1469-0691.12418 (2014).
23 Jarrad, A. M., Karoli, T., Blaskovich, M. A., Lyras, D. & Cooper, M. A. Clostridium difficile drug pipeline: challenges in discovery and development of new agents. Journal of medicinal chemistry 58, 5164-5185, doi:10.1021/jm5016846 (2015).
24 Perkins, H. R. & Nieto, M. The chemical basis for the action of the vancomycin group of antibiotics. Annals of the New York Academy of Sciences 235, 348-363 (1974).
25 Surawicz, C. M. et al. Guidelines for diagnosis, treatment, and prevention of Clostridium difficile infections. The American journal of gastroenterology 108, 478-498; quiz 499, doi:10.1038/ajg.2013.4 (2013).
26 Gallo, A., Passaro, G., Gasbarrini, A., Landolfi, R. & Montalto, M. Modulation of microbiota as treatment for intestinal inflammatory disorders: an uptodate. World J Gastroentero 22, 7186-7202, doi:10.3748/wjg.v22.i32.7186 (2016).
27 Goudarzi, M. et al. Antimicrobial susceptibility of Clostridium difficile clinical isolates in iran. Iran Red Crescent Med J 15, 704-711, doi:10.5812/ircmj.5189 (2013).
28 Adler, A. et al. A national survey of the molecular epidemiology of Clostridium difficile in Israel: the dissemination of the ribotype 027 strain with reduced susceptibility to vancomycin and metronidazole. Diagn Microbiol Infect Dis 83, 21-24, doi:10.1016/j.diagmicrobio.2015.05.015 (2015).
29 Freeman, J. et al. Pan-European longitudinal surveillance of antibiotic resistance among prevalent Clostridium difficile ribotypes. Clin Microbiol Infect 21, 248 e249-248 e216, doi:10.1016/j.cmi.2014.09.017 (2015).
30 Tenover, F. C., Tickler, I. A. & Persing, D. H. Antimicrobial-resistant strains of Clostridium difficile from North America. Antimicrob Agents Chemother 56, 2929-2932, doi:10.1128/AAC.00220-12 (2012).
31 Leeds, J. A., Sachdeva, M., Mullin, S., Barnes, S. W. & Ruzin, A. In vitro selection, via serial passage, of Clostridium difficile mutants with reduced susceptibility to fidaxomicin or vancomycin. J Antimicrob Chemother 69, 41-44, doi:10.1093/jac/dkt302 (2014).
32 Goldstein, E. J., Babakhani, F. & Citron, D. M. Antimicrobial activities of fidaxomicin. Clin Infect Dis 55 Suppl 2, S143-148, doi:10.1093/cid/cis339 (2012).
33 Sebaihia, M. et al. The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nature genetics 38, 779-786, doi:10.1038/ng1830 (2006).
34 Janoir, C. Virulence factors of Clostridium difficile and their role during infection. Anaerobe 37, 13-24, doi:10.1016/j.anaerobe.2015.10.009 (2016).
35 Awad, M. M., Johanesen, P. A., Carter, G. P., Rose, E. & Lyras, D. Clostridium difficile virulence factors: Insights into an anaerobic spore-forming pathogen. Gut Microbes 5, 579-593, doi:10.4161/19490976.2014.969632 (2014).
36 Borriello, S. P., Davies, H. A., Kamiya, S., Reed, P. J. & Seddon, S. Virulence factors of Clostridium difficile. Rev Infect Dis 12 Suppl 2, S185-191 (1990).
37 Davies, H. A. & Borriello, S. P. Detection of capsule in strains of Clostridium difficile of varying virulence and toxigenicity. Microb Pathog 9, 141-146 (1990).
38 Tasteyre, A. et al. A Clostridium difficile gene encoding flagellin. Microbiology 146 ( Pt 4), 957-966, doi:10.1099/00221287-146-4-957 (2000).
39 Ozeri, V. et al. A two-domain mechanism for group A streptococcal adherence through protein F to the extracellular matrix. EMBO J 15, 989-998 (1996).
40 Schneewind, O., Fowler, A. & Faull, K. F. Structure of the cell wall anchor of surface proteins in Staphylococcus aureus. Science 268, 103-106 (1995).
41 Coley, J., Archibald, A. R. & Baddiley, J. A linkage unit joining peptidoglycan to teichoic acid in Staphylococcus aureus H. FEBS Lett 61, 240-242 (1976).
42 Mazmanian, S. K., et al. Sortase-catalysed anchoring of surface proteins to the cell wall of Staphylococcus aureus. Mol Microbiol 40, 1049-1057 (2001).
43 Mazmanian, S. K., Liu, G., Ton-That, H. & Schneewind, O. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285, 760-763 (1999).
44 Marraffini, L. A., et al. Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria. Microbiology and molecular biology reviews : MMBR 70, 192-221, doi:10.1128/MMBR.70.1.192-221.2006 (2006).
45 Ton-That, H., Marraffini, L. A. & Schneewind, O. Protein sorting to the cell wall envelope of Gram-positive bacteria. Biochimica et biophysica acta 1694, 269-278, doi:10.1016/j.bbamcr.2004.04.014 (2004).
46 Spirig, T., Weiner, E. M. & Clubb, R. T. Sortase enzymes in Gram-positive bacteria. Mol Microbiol 82, 1044-1059, doi:10.1111/j.1365-2958.2011.07887.x (2011).
47 Suree, N., Jung, M. E. & Clubb, R. T. Recent advances towards new anti-infective agents that inhibit cell surface protein anchoring in Staphylococcus aureus and other gram-positive pathogens. Mini reviews in medicinal chemistry 7, 991-1000 (2007).
48 Jonsson, I. M., Mazmanian, S. K., Schneewind, O., Bremell, T. & Tarkowski, A. The role of Staphylococcus aureus sortase A and sortase B in murine arthritis. Microbes and infection / Institut Pasteur 5, 775-780 (2003).
49 Newton, S. M. et al. The svpA-srtB locus of Listeria monocytogenes: fur-mediated iron regulation and effect on virulence. Molecular microbiology 55, 927-940, doi:10.1111/j.1365-2958.2004.04436.x (2005).
50 Budzik, J. M. et al. Amide bonds assemble pili on the surface of bacilli. Proceedings of the National Academy of Sciences of the United States of America 105, 10215-10220, doi:10.1073/pnas.0803565105 (2008).
51 Marraffini, L. A. & Schneewind, O. Targeting proteins to the cell wall of sporulating Bacillus anthracis. Molecular microbiology 62, 1402-1417, doi:10.1111/j.1365-2958.2006.05469.x (2006).
52 Claessen, D. et al. A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils. Genes & development 17, 1714-1726, doi:10.1101/gad.264303 (2003).
53 Capstick, D. S., Willey, J. M., Buttner, M. J. & Elliot, M. A. SapB and the chaplins: connections between morphogenetic proteins in Streptomyces coelicolor. Molecular microbiology 64, 602-613, doi:10.1111/j.1365-2958.2007.05674.x (2007).
54 van Leeuwen, H. C. et al. Clostridium difficile sortase recognizes a (S/P)PXTG sequence motif and can accommodate diaminopimelic acid as a substrate for transpeptidation. FEBS Lett 588, 4325-4333, doi:10.1016/j.febslet.2014.09.041 (2014).
55 Schneewind, O., et al. Cell wall sorting signals in surface proteins of gram-positive bacteria. The EMBO journal 12, 4803-4811 (1993).
56 Janulczyk, R. & Rasmussen, M. Improved pattern for genome-based screening identifies novel cell wall-attached proteins in gram-positive bacteria. Infection and immunity 69, 4019-4026, doi:10.1128/IAI.69.6.4019-4026.2001 (2001).
57 Donahue, E. H. et al. Clostridium difficile has a single sortase, SrtB, that can be inhibited by small-molecule inhibitors. BMC Microbiol 14, 219, doi:10.1186/s12866-014-0219-1 (2014).
58 Tulli, L. et al. CbpA: a novel surface exposed adhesin of Clostridium difficile targeting human collagen. Cellular microbiology 15, 1674-1687, doi:10.1111/cmi.12139 (2013).
59 Vollmer, W., Joris, B., Charlier, P. & Foster, S. Bacterial peptidoglycan (murein) hydrolases. FEMS microbiology reviews 32, 259-286, doi:10.1111/j.1574-6976.2007.00099.x (2008).
60 Zong, Y., Mazmanian, S. K., Schneewind, O. & Narayana, S. V. The structure of sortase B, a cysteine transpeptidase that tethers surface protein to the Staphylococcus aureus cell wall. Structure 12, 105-112 (2004).
61 Zhang, R. et al. Structures of sortase B from Staphylococcus aureus and Bacillus anthracis reveal catalytic amino acid triad in the active site. Structure 12, 1147-1156, doi:10.1016/j.str.2004.06.001 (2004).
62 Yin, J. C. et al. Structural Insights into Substrate Recognition by Clostridium difficile Sortase. Front Cell Infect Microbiol 6, 160, doi:10.3389/fcimb.2016.00160 (2016).
63 Jacobitz, A. W. et al. Structural and computational studies of the Staphylococcus aureus sortase B-substrate complex reveal a substrate-stabilized oxyanion hole. J Biol Chem 289, 8891-8902, doi:10.1074/jbc.M113.509273 (2014).
64 Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126-2132, doi:10.1107/S0907444904019158 (2004).
65 Collaborative Computational Project, N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50, 760-763, doi:10.1107/S0907444994003112 (1994).
66 Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 58, 1948-1954 (2002).
67 Hu, P., Huang, P. & Chen, M. W. Curcumin reduces Streptococcus mutans biofilm formation by inhibiting sortase A activity. Arch Oral Biol 58, 1343-1348, doi:10.1016/j.archoralbio.2013.05.004 (2013).
68 Maresso, A. W. et al. Activation of inhibitors by sortase triggers irreversible modification of the active site. J Biol Chem 282, 23129-23139, doi:10.1074/jbc.M701857200 (2007).
69 McAllister, K. N. et al. Using CRISPR-Cas9-mediated genome editing to generate C. difficile mutants defective in selenoproteins synthesis. Sci Rep 7, 14672, doi:10.1038/s41598-017-15236-5 (2017).
70 Jyun-Cyuan Chang. Molecular Analysis of Substrate Specificity of Clostridium Difficile Sortase. National Cheng Kung University Department of Microbiology & Immunology, (2017).