槲皮素抑制2型猪链球菌SrtA的效力及分子机制研究

Drug-resistance of Streptococcus suis (S. suis) becomes increasingly serious with the overuse of antibiotics in infection control. To address this problem, people turn to looking for the anti-infection drugs which target to key virulence factors of pathogens. Sortase A (SrtA) is not necessary to growth, thus its targeting drug does not lead to drug-resistance. Furthermore, it plays important roles in anchoring many key virulence factors onto cell wall of gram-positive pathogen. These features made SrtA an ideal target in anti-infection. It has been revealed that SrtA inhibitors have great potency in anti-infection of several pathogens. However, no research about S. suis SrtA and its inhibitors has been reported. Recently we identified a S. suis SrtA inhibitor quercetin, a natural product which displays strong inhibition on SrtA activity from S. suis serotype 2. In this study, by measuring SrtA activity and the anchoring of its substrate protein on cell wall, the inhibitory efficacy of quercetin on S. suis SrtA will be determined. Moreover, by resolving 3D-structure of S. suis SrtA and molecular docking of quercetin, the binding sites between SrtA and quercetin are going to be determined and confirmed by site-directed mutations. Altogether, the potential of quercetin in anti-infection of S. suis will also be evaluated. This study will shed new light on the development of new drugs targeting SrtA against S. suis infections.

随着猪链球菌病防治过程中对抗生素的过度依赖，猪链球菌耐药性问题日益突出。针对耐药性这一问题，靶向重要病原菌毒力的抗感染药物成为重要选择。转肽酶A（SrtA）不为细菌生长所必需，其靶向药物不会引起耐药性，而且它介导很多重要毒力因子定位到细胞壁上，使其成为一个理想的抗感染药物靶标。SrtA抑制剂在抗某些病原菌感染方面已经显示出很大潜力，但是目前还没有猪链球菌SrtA及其抑制剂的报道。前期我们筛选到一种抑制2型猪链球菌SrtA的天然产物槲皮素，初步结果显示其效力强于多数已知的SrtA抑制剂。本项目拟通过检测SrtA活力及其底物蛋白在细菌表面的定位，确定槲皮素对SrtA的抑制效力。通过解析SrtA的三维结构，进行与槲皮素的分子对接，分析SrtA与槲皮素的结合位点并定点突变验证，阐明其抑制机制。总体评估槲皮素抗猪链球菌感染的潜力。这些研究将为以SrtA为靶标的新型抗猪链球菌感染药物开发提供理论基础。

长期以来，转肽酶A（SrtA）是一个理想的抗革兰氏阳性病原菌的药物靶标。虽然猪链球菌是一种重要的人兽共患病原菌，但是缺少对猪链球菌转肽酶SrtA（Ss-SrtA）的研究报道。本文不仅对转肽酶Ss-SrtA的酶学性质进行研究，而且评价天然产物对转肽酶Ss-SrtA的抑制效力。在pH 6.0-7.5、45℃的条件下，转肽酶Ss-SrtA发挥最大的酶活力。转肽酶Ss-SrtA对底物abz-LPATG-dnp的Km值为6.7μM。与金黄色色葡萄球菌SrtA（Sa-SrtA）依赖Ca2+不同，转肽酶Ss-SrtA是不依赖Ca2+的。进一步的蛋白结构预测发现，转肽酶Ss-SrtA的第111号赖氨酸和第180号天冬氨酸形成盐桥，取代了转肽酶Sa-SrtA中Ca2+的功能以便稳定底物结合裂缝。此外，蛋白氨基酸点突变的方法证实H126，C192和R200是转肽酶Ss-SrtA的关键性氨基酸残基。为了测定天然产物对转肽酶Ss-SrtA的抑制效力，还开展了有潜力抑制剂的研究。首次发现毛蕊花糖苷、异槲皮素和黄芪苷是转肽酶SrtA的抑制剂，IC50值分别为36.3±1.3μM，100.0±1.3μM和85.4±1.5μM。基于FITC标记底物设计的内源性转肽酶Sa-SrtA的酶活性测定方法，结果显示毛蕊花糖苷、异槲皮素和黄芪苷分别抑制了转肽酶Sa-SrtA的86%，28%和45%酶活力。总之，本研究阐明了转肽酶Ss-SrtA的酶学性质，预测了转肽酶Ss-SrtA的非Ca2+依赖性催化机制和发现了转肽酶Ss-SrtA的潜在抑制剂。