环D-脯氨酸D-苯丙氨酸对金黄色葡萄球菌的受体定位及对能量代谢影响

An active antimicrobial of Alcaligenes sp. was isolated and identified as cyclo(D)-Pro-(D)-Phe by high throughout screening model, which belongs to Cyclic dipeptides. It was also discovered spanning activities as antibacterial. Therefore, this compound would hopefully developed for clinical applications and preservatives. But the essential of its antibacterial is still unclear, its binding receptors is not reported, its regulated transcriptors and metabolism route is invisible. These cause its signal transduction darkness. Consequently we will study the nature of its microbe inhibiting. First, After some inquiry through Cheminformatics, it will be Quantitative calculation in order to acknowledge its physical-chemical property and affinity proteins. Then its receptors for Staphyloccocus aureus will be found through 2-DE SDS-PAGE and ligand blotting analysis. Finally, the signaling pathway would be evaluated by comparing proteomics results, and their target and bacteriostasis mechanism will be uncovered. All above efforts will form a theoretic and practical basis for new peptide antibiotics and bio-preservative.

我们通过建立高通量筛选模型，成功从一株产碱杆菌（Alcaligenes sp.）分离到一种高活性广谱抗菌成分环二肽，即环D-脯氨酸D-苯丙氨酸(cyclo(D)-Pro-(D)-Phe)。它具有抗菌等广泛的生理活性,具备成为新型抗生素，防腐剂及免疫药物的优良品质。但是环二肽以何种机制抑制细菌至今还不清楚，其直接作用受体也未见报道，对其调控的蛋白及代谢所知甚少，导致其信号传导通路无法确认。本研究拟通过化学信息学，了解其毒理学性质并预测其受体蛋白；利用蛋白双向电泳、配体印迹分析实验探明其对金黄色葡萄球菌的作用受体；结合蛋白组学阐明其最适靶蛋白及对能量代谢的影响，从而推测其信号传导通路。为开发新型抗生素和生物食品防腐剂提供相应的理论依据。

环(苯丙氨酸脯氨酸)(cFP)是一种细胞外群体感应信号分子，能够根据细菌密度，协调细菌应对外部应激。本课题采用iTRAC法分析cFP对金黄色葡萄球菌 (S. aureus) CGMCC 1.1861蛋白表达的影响进行分析和定量。结果表明，cFP对高温、酸碱、紫外光和荧光均保持稳定。此外，S. aureus蛋白质组中鉴定出1296种蛋白质。22种蛋白质与双组分调控系统(TCRS)有关。差异质谱分析显示，cFP仅上调43个蛋白，下调41个蛋白。其中氨基酸代谢、脂质代谢、辅助因子和维生素代谢途径差异最大。结果表明，cFP可下调毒力因子，上调脂质和氨基酸代谢，促进乙酰化和磷酸化。同时，利用AutoDock4.2.6软件计算分析cFP与潜在受体之间的模拟结合。cFP与AgrA (Q2FZW6)的结合能为- 2.95 Kcal/mol，结合位点为ASN211。理论计算表明，主要的群体感应(QS) AgrC/AgrA系统可以自动与cFP结合。因此，cFP可能作为一种竞争性抑制剂，阻遏AgrC/AgrA (AgrA家族)信号转导。验证试验表明cFP对乳酸脱氢酶(LDH)活性无明显影响。但处理4、6 h后，金黄色葡萄球菌ATP浓度降低，2 h后醇脱氢酶(ADH)活性降低。iTRAC实验中共有12个显著差异表达蛋白(DEP)与信号转导有关。其中，Rot (Q9RFJ6)和能够抑制Agr系统转录的SarR (Q9F0R1)上调，而ESAT-6蛋白A (Q2G189)、苯酚可溶性调节素Psm(Q2FZA4)、肽ABC转运体渗透酶(Q2G168)等下调。同时cFP下调金黄色葡萄球菌AgrA (Q2FWM4)，表明cFP可能对Agr系统有抑制作用。

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