SurA影响大肠埃希菌持留菌形成及存活的分子机制

Certain infectious diseases caused by pathogenic bacteria are typically chronic in nature. Chronic infections are recalcitrance to treatment with antibiotics in clinical, and consequently follow up with the problems of recurrence infection, drug-resistance and bacterial bio-films. Recently, studies have attributed these clinical problems with persister which is a small fraction of multi-drug tolerance bacteria. But, the mechanism of persister formation, especially the related key molecular, eluded us for a very long time. In our previous studies, we found that E. coli ΔsurA, surA knockout strain, was more susceptible to stresses than the wild type strain E. coli BW25113 and the other gene knockout strains, and its mutant phenotypes could be complemented by surA, which showed that surA was related to persister. SurA is one of periplasmic chaperones, which assists the biogenesis, assembly and transit of outer membrane proteins. However, how does SurA involve in the formation and maintance of persisters is not clear. In this study, we try to use SurA-specific photocrosslinker, in combination with proteomics and gene knockout technologies, to explore the target of SurA，the effect of SurA on persister as chaperone and toxin. SurA might be an effective target for drug against persister, and the results of this research would lay a foundation of the research in chronic infections.

持留菌是慢性感染复发、细菌耐药以及生物膜形成等临床治疗难题的根本原因。然而，持留菌的形成机制和影响持留菌形成与存活的关键分子尚不明确。我们前期发现大肠埃希菌surA基因缺失菌株（E. coli ΔsurA）持留菌的形成和存活能力明显小于野生菌株E. coli BW25113和其他基因缺失菌株，而且该菌株的持留能力在回补surA基因后恢复到野生菌株的水平，提示surA与持留菌的形成相关。SurA是细菌周质中的伴侣分子，参与外膜蛋白的合成、组装及转运，但其在持留菌形成中的作用尚没有研究报道。本项目拟运用SurA蛋白特异性光交联探针、结合蛋白质组学及基因敲除等技术手段，研究在持留菌形成过程中SurA的靶蛋白、其伴侣作用对靶蛋白的保护效应以及毒素-抗毒素原件效应，从而揭示SurA影响持留菌形成与存活的分子机制。SurA可能会成为抗持留菌药物的靶点，本研究对慢性感染的机制研究和药物设计奠定基础。

持留菌是细菌群落中代谢停滞且对致死剂量抗生素具有耐受性的细菌，是慢性感染难以治愈的根本原因。持留菌形成和存活的机制非常复杂，目前其确切的机制仍然不是很清楚。本课题以大肠埃希菌持留菌为研究对象，基于大肠埃希菌单基因敲除文库（KEIO突变体文库）的高通量筛选，研究细菌持留的关键基因及分子机制。主要研究内容及结果如下：（1）筛选持留菌形成与存活的重要基因并进行初步验证。我们筛选到18个突变株(ΔacrA, ΔacrB, ΔddlB, ΔdnaG, ΔgltI, ΔhlpA, ΔlpcA, ΔrecG, ΔrecN, ΔrfaH, ΔruvC, ΔsurA, ΔtatC, ΔtolQ, ΔuvrD, ΔxseA和ΔydfI)不能形成对抗生素的持留；其中，ΔgltI, ΔhlpA, ΔruvC, ΔddlB, ΔydfI 和 ΔtatC对抗生素持留具有特异性。此外，编码细菌周质蛋白的基因突变体(ΔsurA, ΔlpcA, ΔhlpA和ΔgltI）对抗生素的持留性降低比其它鉴定出的突变体更为明显，其中以ΔsurA和ΔlpcA突变体表现最为突出。（2）明确关键基因surA及lpcA对持留菌形成与存活的重要性。在体外的细菌持留模型中，与大肠埃希菌野生株BW25113相比，surA和lpcA突变体对多种抗生素、酸性、高渗和高温等“压力”因素的持留能力降低了至少100-1000倍。在体内的细菌持留模型中（小鼠感染模型），surA和lpcA突变体的持留性和致病性均低于尿路致病性大肠埃希菌野生菌株UTI89。结果表明，体外筛选出的耐托舒沙星的大肠埃希菌持留菌相关基因surA和lpcA在体内也得到验证。（3）surA参与持留菌形成与存活的作用机制。SurA（0.5μg/mL）对大肠埃希菌具有毒素效应，而对痢疾志贺菌、伤寒沙门菌和金黄色葡萄球菌没有毒性，主要通过下调细菌多个代谢通路引起细菌死亡。此外，SurA通过分子伴侣效应，影响多个重要膜蛋白的合成。该研究成果明确了SurA在细菌持留中的关键作用，阐明了SurA通过其毒素效应下调代谢及通过膜蛋白的合成影响细菌的持留及致病力，为大肠埃希菌的持留机制提出了新的见解，为大肠埃希菌持留菌疫苗和药物的研究奠定基础。