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標題: | 表皮葡萄球菌TcaR與TcaR-抗生素複合體之晶體結構及作用機制 Structural study of TcaR and its complexes with multiple antibiotics from Staphylococcus epidermidis |
作者: | Yu-Ming Chang 張育銘 |
指導教授: | 王惠鈞 |
關鍵字: | 表皮葡萄球菌,生物膜,抗生素,操縱子,阻抑蛋白, TcaR,Staphylococcus epidermidis,Biofilm,antibiotics,EMSA, |
出版年 : | 2010 |
學位: | 博士 |
摘要: | 葡萄球菌屬(Staphylococcus)共有32種與8亞種,其中寄宿於人類皮膚上的表皮葡萄球菌(Staphylococcus epidermidis) 為重要的革蘭氏陽性條件致病菌,日常對健康並無影響,但對於免疫力低的病人,卻會造成嚴重感染,導致敗血症、人工瓣膜心內膜炎、骨髓炎、化膿性關節炎及各種異物感染,甚至致命。目前全球醫療機構皆鎖定表皮葡萄球菌與金黃色葡萄球菌(S. aureus)為院內感染之首號菌種,而隨著抗生素的廣泛使用,其抗藥性也不斷增強,成為臨床醫療的棘手問題。在之前的相關研究指出,TcaR是ica操縱子的阻抑蛋白,此操縱子會影響表皮葡萄球菌生物膜的形成,進而影響其抗藥性。在本篇論文中,我們解析了TcaR以及TcaR-抗生素複合體之晶體結構,並發現抗生素的結合會造成TcaR上的DNA結合部位結構明顯改變,此變化為表皮葡萄球菌因應抗生素入侵而啟發生物膜合成之重要關鍵。此外,我們也明確地找出TcaR結合在ica操縱子上的特定序列,並利用EMSA實驗證實抗生素等化學物質能抑制TcaR與ica操縱子之結合。最後,我們更進一步進行in vivo實驗,確定抗生素能誘發表皮葡萄球菌大量增生生物膜之抵禦機制。總結來說,我們的研究不僅明確將TcaR之抗生素結合位置確實定位,並解開表皮葡萄球菌如何調控生物膜生成之機制,以幫助未來解決葡萄球菌抗藥性此棘手問題。 There are 32 species and 8 sub-species of Staphylococcus. They cause a wide range of diseases in humans and animals such as skin infections, pneumonia, food poisoning and blood poisoning. Among them, S. epidermidis is the sister species of S. aureus which often causes infection in immunocompromised individuals. It produces biofilm to protect itself from host immune system and enhance their resistance to antibiotic chemotherapy. Antibiotic resistance is an increasing problem throughout the developed world. An understanding of the mechanism of their regulation is urgently needed for efficient treatment of bacterial infections. It has been known that TcaR is a negative regulator of transcription of the ica locus and prevents the poly-N-acetylglucosamine (PNAG) production and biofilm formation in Staphylococcus epidermidis. Although TcaR was shown to interact with the ica promoter, the precise binding region and the mechanism of interaction remained unclear. Here we present the 3D structure of TcaR in its apo form and in complex with salicylate as well as several aminoglycoside and β-lactam antibiotics. A comparison of the native and complex TcaR structures indicates that the mechanism of regulation involves a large conformational change in the DNA binding lobe. In addition, we deduced the consensus binding sequence of two [~TTNNAA] hexamers embedded in a 16 bp sequence for a TcaR dimer. The results of the electrophoretic mobility shift assay confirmed that six TcaR dimers bind specifically to three ~33 bp segments that are close to the IcaR binding region with varying affinities, and its repressor activity would be interfered by salicylate and multiple antibiotics. Moreover, our study has also delineated the antibiotics binding site of TcaR and confirmed that the antimicrobial compounds we tested were shown not only to inhibit TcaR-DNA interaction but also to further induce biofilm formation in S. epidermidis in our in vivo assay. In summary, we discover for the first time a MarR family protein can interact directly with different classes of natural antimicrobial compounds to affect its repressor activity, and the crystal structures of TcaR complexes highlight the importance and versatility of regulatory systems in bacterial antibiotic resistance. We hope these results could provide further understanding for the effect of antibiotic exposure on bacterial antibiotic resistance through biofilm formation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22602 |
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顯示於系所單位: | 生化科學研究所 |
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