探討抗甲氧苯青黴素金黃色葡萄球菌之潛力抗菌藥與膜蛋白YidC2的交互作用

Abstract

在現代社會中，由於抗生素的濫用而使得越來越多具有抗藥性的細菌出現，其中之一為抗甲氧苯青黴素金黃色葡萄球菌，被此菌感染嚴重會致死，令人擔心的是這種細菌對目前市面上大部分的抗生素都具有抗藥性，以至於治療困難，所以發展新穎抗菌藥也成為科學家們重要的課題。在先前研究發現非固醇類抗發炎藥物希樂葆 (celecoxib)衍生物Cpd36及Cpd46能有效抑制抗甲氧苯青黴素金黃色葡萄球菌的生長，並利用次世代定序(NGS)找出膜蛋白YidC2可能參與了抗菌機制之中。因此，我們想確認是否因YidC2與化合物Cpd36、Cpd46之間有交互作用，而影響金黃色葡萄球菌的生存。從我們的實驗結果中發現增加細菌中YidC2的表現量會使得細菌對Cpd46更具有抗性。除此之外，我們利用YidC2移位酶的受質三磷酸腺苷合成酶次單元c 分析YidC2的功能，結果發現在Cpd46存在的環境中受質無法順利移位到細胞膜上，因此而導致三磷酸腺苷無法正常合成。我們也發現在施加Cpd46時，會使得細菌YidC2表現量上升，但是這些YidC2無法幫助其受質再次移位到細胞膜上。綜合上述結果我們認為Cpd46可能直接結合在YidC2上。為了確認Cpd36和Cpd46是否與YidC2蛋白直接結合，我們利用生物物理的方式分析熱力學及動力學數據的變化，可惜的是，礙於化合物溶解度以及蛋白質特性等因素，無法從數據中看到直接結合的反應。根據我們的研究，膜蛋白YidC2確實在Cpd46的殺菌活性中扮演一個重要角色，且YidC2為一個極具潛力的作用目標用以發展新抗菌藥物，來解決抗甲氧苯青黴素金黃色葡萄球菌所造成的問題。

Due to inappropriate use of antibiotics, the emergence of antimicrobial-resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Streptococcus pneumonia, has been a critical problem nowadays. In particular, infections of MRSA, resistant to most antibiotics, often cause a higher mortality in patient. In our previous study, two celecoxib derivatives, Cpd36 and Cpd46, were identified to exhibit inhibitory activity against MRSA. Subsequent mutation analysis of S. aureus resistant isolates by next-generation sequencing identified a membrane protein translocase, YidC2, as the potential target of these compounds. Here, we aim to investigate the interaction between the YidC2 and Cpd36 and Cpd46. The results showed that ectopic expression of YidC2 can decrease the susceptibility of S. aureus to Cpd46. The level of ATP synthase subunit c (ATPsc) on bacterial membrane were also reduced by Cpd46 treatment, leading to a reduction of ATP level in bacteria. Although YidC2 protein level in bacteria was increased with exposure to Cpd46, the raised YidC2 level didn’t reverse the reduction of ATPsc translocation. To further investigate whether Cpd46 bind to YidC2 directly, recombinant YidC2 was prepared by overexpressing protein in E. coli followed by extraction with detergent. Next, to explore the interaction between YidC2 and compounds, biophysical approaches were used to monitor the change of several important physiochemical parameters, including enthalpy, entropy, free energy and the thermodynamic profile. However, no significant biophysical evidences showed these compounds binding to YidC2 directly. Altogether, our results showed that YidC2 plays an important role in the antibacterial activity of Cpd46, and YidC2 is a potential target for new anti-MRSA drugs development.