困難梭狀桿菌(Clostridium difficile)為革蘭氏陽性、產孢子之厭氧桿菌且為具有多重抗藥性之人類致病菌。其主要發生於醫院及照護中心中使用抗生素治療、免疫力低下的病人。由於抗生素治療會破壞宿主腸道內正常菌叢，使得困難梭狀桿菌得以增生並分泌毒素。近年來，因抗生素使用型態的改變，導致抗藥性及高致病性菌株的出現，又目前尚未有適當治療方式避免疾病復發，因此其重要性與日俱增。若細菌欲感染宿主，首先細菌須定殖於宿主細胞表面，由於細菌表面具有許多附著分子及毒力因子會與宿主細胞產生交互作用，進而附著及入侵細胞，最終感染宿主導致疾病的產生。而這些附著因子及毒力因子可藉由分選酶(Sortase B)將其錨定於細菌細胞壁上，因此SrtB為一與致病相關之重要因子。本篇研究著重於探討源自困難梭狀桿菌的SrtB的結晶結構，同時藉由尋找合適的分選酶抑制劑，欲透過已知抑制劑結構加上SrtB的結構進一步設計新的藥物來治療疾病。在本篇研究中，藉基因重組的方法將困難梭狀桿菌的SrtB基因表現於大腸桿菌中，並藉親和性膠體層析法純化重組蛋白。SrtB蛋白質晶體結構顯示，SrtB包含三個α-螺旋體(α-helix)、兩個310-螺旋體(310-helix)及八個β-鏈(β-strand)且以單體的形式存在於溶液中。我們進一步將困難梭狀桿菌的SrtB的結晶結構與炭疽桿菌、金黃葡萄球菌及化膿性鏈球菌之SrtB相互比較，雖整體結構是相似的，但與酵素作用相關之Cys-His-Asp催化環(Cys-His-Asp triad)在結構型態上有所不同。此外，我們也成功獲得SrtB deltaN26,C209A-PPKTG的複合物晶體。目前尚未有SrtB與其受質之作用機制被發表，因此若能得知複合物晶體的結構將有利於探討酵素與其受質間之交互作用。此外，螢光共振能量轉移(FRET)結果顯示，薑黃素與AAEK1能有效抑制困難梭狀桿菌的SrtB的活性。綜合而論，我們解出困難梭狀桿菌的SrtB的晶體結構且找到薑黃素及AAEK1為有效之SrtB抑制劑。後續我們將優化長晶條件並透過SrtB deltaN26,C209A-PPKTG的複合物晶體結構，進一步在分子層級研究困難梭狀桿菌SrtB作用分子機轉，此外，我們希望能以結構為基礎設計SrtB抑制劑，其不僅能抑制SrtB的活性還能降低細菌產生抗藥性的機率，此將有利於治療困難梭狀桿菌感染之患者。

Clostridium difficile (C. difficile) is a Gram-positive, spore-forming, and multidrug-resistant bacterial pathogen. C. difficile infection mostly occurs in hospitalized patients treated with antibiotic therapies. Antibiotic treatments damage host beneficial bacteria allowed C. difficile has chance to proliferate and exotoxin secretion result in Clostridium difficile-associated disease (CDAD). Recently, increasing numbers of drug-resistant and highly pathogenic strains of C. difficile were found due to the administration of broad-spectrum antibiotics. Therefore, there is an urgent need to develop new targets to control the diseases. Cell surface proteins, which play many important roles in pathogenesis are the potential targets. However, those surface proteins were anchored to the cell surface by sortase, a cysteine protease, mainly exists in Gram-positive bacteria and mediates surface proteins forming covalent bond with bacterial cell wall via a transpeptidation reaction. Thus, sortases can be considered as a promising target for the development of inhibitory molecules to block the surface proteins display in cell wall. In this study, I have successfully determined the crystal structure of an anchored protein, sortase B, from C. difficile and aim to use the crystal structure of sortase for future structure-based drug design against CDAD through inhibition the protease activity of SrtB. Recombinant C. difficile SrtB deltaN26 protein was successfully expressed in Escherichia coli and purified by Ni2+-NTA affinity chromatography. C. difficile SrtB deltaN26 is characterized to form monomer in solution. The crystal structure of SrtB deltaN26 was solved to 2.8 Å and consisted of three α-helices, two 310-helices and eight β-strands. The overall structure of C. difficile SrtB deltaN26 is similar to these of B. anthracis, S. aureus, and S. pyogenes. However, the orientations and distances between catalytic residues of Cys-His-Asp catalytic triad are different, suggesting the binding-specificity of SrtB in C. difficile. Due to scarcity of crystal structure of sortase-substrate complex in the present literature, the molecular mechanism of how sortase anchoring the surface protein to cell wall remains to be explored. Therefore, I have crystallized SrtB deltaN26,C209A-PPKTG complex for future structural determination. Furthermore, I discovered that curcumin and AAEK1 were effective inhibitors of C. difficile SrtB deltaN26 by fluorescence resonance energy transfer (FRET)-based assay. In summary, results from my thesis studies have provided the atomic resolution of C. difficile SrtB, identified the effective inhibitors and crystallized SrtB deltaN26,C209A-PPKTG complex. My studies have contributed to the framework toward understanding the mode of actions of sortase in Gram-positive bacteria and as a first step for the development of marketable new drug against CDAD.

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