困難梭狀桿菌Clostridium difficile (C. difficile)為革蘭氏陽性、厭氧桿狀菌、並且會產孢子。在醫院中因為抗生素的大量使用，會導致宿主腸道內正常菌叢被破壞。當宿主腸道內的菌叢被破壞後，會使得C. diffcile 更容易附著在宿主腸道內，並且複製。進而引發發炎反應，導致C. diffcile相關疾病的發生(CDAD)。近年來，抗藥性和高度致病性的菌株增加已經成為主要的問題，但目前對於C. diffcile 感染上還缺乏有效的治療。因此，我們需要發展替代性的治療方法，希望能有效控制疾病並且減少病菌產生抗藥性。分選酶(Sortase)為cysteine protease，存在於Gram-positive的細菌中，並且可以將含有毒力因子的表面蛋白嵌入至細菌的細胞壁上，使細菌具有感染能力。Sortase可視為替代性的治療目標，發展抑制劑減緩病菌的感染症狀。C. diffcile只帶有一個sortase，又稱為sortase B (Srt B)，C. diffcile的sortase B可以辨認具有(S/P)PXTG序列的表面蛋白，並且催化切割Threonine和glycine之間。在不同種的革蘭氏陽性菌中，sortase B有高度的結構相似性。然而在受質(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sortase B蛋白的結晶結構。由於sortase-substrate complex的結構目前仍了解的有限。因此，sortase在受質特異性(substrate specificity)的相關機制，目前仍是不清楚。在之前的研究中發現，sortase上的6-7 loop在substrate的辨認上扮演很重要的角色。除此之外，將金黃色葡萄球菌分選酶A (S. aureus Sortase A)的6-7 loop置換成金黃色葡萄球菌分選酶B (S. aureus Sortase B)的loop，可以改變substrate specificity的形式。我們實驗室也成功模擬出C. difficile SrtB-PPKTG complex的模型。我們比較已知的S. aureus SrtB-NPQT* complex結構和我們C. difficile SrtB-PPKTG complex 模型，在6-7 loop結構表面上的residue。為了瞭解6-7 loop在C. difficile SrtB所扮演的角色。我採用了定位突變(site-directed mutagenesis)、螢光共振能量轉移測定(fluorescence resonance energy transfer) (FRET)、表面等離子共振(surface plasmon resonance) (SPR)去測定這些residue是否參與substrate的辨認。總結而論，我的研究已經顯示6-7 loop在C. difficile SrtB對於substrate辨認上的重要性，並且有利於我們了解分選酶和受質之間的特異性(sortase-substrate specificity)，進而用於發展新的藥物來治療C. difficile相關的疾病。

Clostridium difficile (C. difficile) is a Gram-positive, spore forming, and obligate anaerobic bacteria. C. difficile-associated disease (CDAD) is caused by disruption of host microflora through broad-spectrum antibiotic use. The increased numbers of drug-resistant and highly pathogenic strains of C. difficile are major problems nowadays. Therefore, it is necessary to develop alternative therapeutic targets to control the disease. Sortase is a cysteine protease that anchors surface proteins containing virulent factors to cell wall. Thus, sortase is considered for development the inhibitors to block the virulent factors displaying in cell surface. C. difficile has only one sortase (SrtB). For C. difficile, surface proteins containing (S/P)PXTG motif are recognized by SrtB and cleaved between threonine and glycine. The structures of SrtB are highly similar in different Gram-positive bacteria. However, SrtB recognizes different substrates consisting of distinct sorting sequences. Recently, our lab has successfully determined the crystal structure of SrtB protein from C. difficile. Due to the limited available structures of sortase-substrate complex, the molecular mechanism of how sortases achieve substrate specificity remains unclear. In previous studies, the beta6-beta7 loop of sortase plays a critical role in substrate recognition. Moreover, the beta6-beta7 loop in S. aureus SrtA replacing to the correlated loop from S. aureus SrtB can change the substrate specificity profile. Our lab has published a computational model of C. difficile SrtB-PPKTG complex. Thus, we compared the surface-exposed residues of beta6-beta7 loop with that of S. aureus SrtB-NPQT* complex. To assess the role of beta6-beta7 loop in C. difficile SrtB, I have employed the techniques of site-directed mutagenesis, fluorescence resonance energy transfer (FRET)-based assay and surface plasmon resonance (SPR) to identify the residues involved in the substrate cleavage and binding. In summary, my studies have revealed the important role of beta6-beta7 loop of C. difficile SrtB in substrate recognition and contributed to better understanding in sortase-substrate specificity for development of new drugs against CDAD.

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