头花蓼内生菌抗尿路感染耐药菌化学小分子的发现及其作用机制研究

Polygonum capitatum is an ethnic Miao's characteristic herb in Guizhou province with proven therapeutic effect against urinary tract infection and its drug-resistant bacteria. Due to the generation of drug-resistant bacteria from abuse of antibiotics, the clinical curative effect of urinary tract infection often declined. The close relationship between metabolites of endophytes and their hosts suggested that it is of particular importance to search for new and active compounds from endophytes of P. capitatum for the treatment of resistant urinary tract infections. Previous efforts has resulted in the discovery of two strains of endophytes of P. capitatum whose metabolites showed good inhibitory activity against the resistant bacteria of urinary tract infection. Chemical analysis indicated that the two endophytes and P. capitatum itself shared some same metabolites, but differences were also found between the two endophytes as well as the endophytes and P. capitatum. In this project, collection of a wider range of endophytes from different aeres of P. capitatum will be carried out for the screening of more active endophytes against resistant strains of urinary tract infection. The metabolites of the active endophytes will then been analyzed by UHPLC-DAD-Q/TOF-HRESIMS/MS, and the compounds with unreported structures will be deemed the target molecules for isolation and structural elucidation. The anti- resistance mechanisms of the active compounds will be investigated by using methods of molecular biology such as RT-PCR and polyacrylamide gel electrophoresis.

头花蓼是贵州特色苗药，对尿路感染及其耐药菌疗效突出。由于抗生素的滥用导致尿路感染产生耐药菌，使常用抗生素的临床疗效普遍降低。内生菌与宿主的代谢产物有着密切联系，从头花蓼内生菌的代谢产物中发现抗（或协同抗生素抗）耐药菌的活性化合物，对治疗耐药菌引起的尿路感染具有重要意义。前期研究已发现2株头花蓼内生菌的代谢产物具有抗尿路感染耐药菌活性，且这些内生菌与头花蓼既有结构相同或相关的代谢产物，内生菌与头花蓼以及内生菌之间也存在明显不同的代谢物质。本项目拟通过广泛采集贵州不同生长条件下头花蓼的内生菌，以尿路感染耐药菌为指示菌，筛选出更多的活性内生菌株；通过UHPLC-DAD-Q/TOF-HRESIMS/MS分析和现代分离、鉴定手段，从其代谢物中获得具有抗尿路感染耐药菌活性的新型化合物；利用RT-PCR及SDS-PAGE等分子生物学技术探索活性化合物的作用机制。

细菌是尿路感染的主要致病原因，然而耐药性的日益突出以及新型抗生素的匮乏严重威胁着人类的生命健康。头花蓼对尿路感染及其耐药菌疗效突出。鉴于内生菌与宿主的代谢产物有着密切联系，从头花蓼内生菌的代谢产物中发现抗（或协同抗生素抗）耐药菌的先导化合物，对治疗尿路感染具有重要意义。本课题从头花蓼内生菌中筛选到抗耐药菌活性优于或等于其宿主头花蓼代谢产物的菌株13株，活性MIC值范围在3.125～200 mg/mL。经分离纯化、活性评估发现化合物镰刀菌酸对多重耐药菌抑制活性较好。并以此为先导化合物，进行结构修饰，获得镰刀菌酸（FA）衍生物41个。结果显示，衍生物5-(4-butylphenyl)picolinic acid (4-BPP)和5-(4-isobutylphenyl)picolinic acid (4-IBPP)对抗多重耐药菌的活性与FA相当。但对普通型铜绿杆菌、大肠杆菌、金黄色葡萄球菌的抗菌活性皆优于先导化合物FA。初步机制研究发现，4-BPP和4-IBPP通过破坏细菌生物膜形成从而对金黄色葡萄球菌以及MASA起到抑菌/杀菌作用。衍生物4-butylpicolinic acid对多重耐药大肠杆菌的MIC为64 μg/mL（0.35 mM），接近于左氧氟沙星的MIC（64μg/mL，0.17 mM），而优于头孢曲松钠（MIC为1024 μg/mL，1.71 mM）。衍生物5-(decyloxy)picolinic acid、5-(dodecyloxy) picolinic acid （5DP）和5-(tetradecyloxy)picolinic acid （5TP）具有协同左氧氟沙星抗多药耐性大肠杆菌效果，使左氧氟沙星的MIC降低4～16倍。同时5DP和5TP具有协同左氧氟沙星抗耐药金黄色葡萄球菌效果，使左氧氟沙星MIC降低4倍。该研究结果对抗耐药菌新药的发现奠定基础。