SdiA-sRNA信号通路对耐药基因接合性转移的调控作用研究

Bacterial conjugation is the transfer of genetic material (plasmid) between bacterial cells by a bridge-like connection, which plays a key role in vast spread of antibiotic resistance genes among clinical pathogens. However, the mechanism underlying conjugation remains largely unknown. We recently found the important functions of quorum sensing (QS) signaling molecule HSL and its receptor SdiA on conjugation by employing E. coli-P. aeruginosa conjugation model, subsequent bioinformatics analysis indicated that several small non-coding RNA (sRNA) that targeted conjugation-associated genes may be transcriptionally regulated by SdiA, suggesting the presence of “HSL-SdiA-sRNA-target genes” signal pathway that regulated conjugation. With these findings as background, this project will focus on the donor cells E. coli to further investigate: 1) identifying the sRNA regulated by SdiA; 2) elucidating the role of SdiA-regulated sRNA in conjugation by gene over-expression and gene knock-out techniques; 3) clarifying the molecular mechanisms underlying the modulation of conjugation-associated genes by sRNA; 4) observing the expression and function of candidate sRNA in HSL-regulated conjugation. The results obtained will not only provide new insight into an important contribution of“HSL-SdiA-sRNA-target genes” to the networks that regulate conjugation, but also provide the molecular basis to develop new strategies based on quorum sensing and sRNA for preventing antibiotic resistance.

接合是细菌间通过性菌毛传递DNA导致耐药基因水平转移的主要方式，但其调节机制尚不十分清楚。我们前期研究发现群体感应系统（QS）信号分子HSL及其受体SdiA具有调控大肠埃希菌-铜绿假单胞菌接合反应的重要功能，进一步采用生物信息学分析发现SdiA可能转录调控多个靶向接合相关基因的非编码小RNA（sRNA）。因此，我们推测存在“HSL-SdiA-sRNA-靶基因”信号通路调控接合反应，是耐药基因水平转移的重要机制。本课题拟通过：①鉴定大肠埃希菌中SdiA调控的sRNA；②过表达和基因敲除候选sRNA，明确其对接合反应的调控作用；③探究候选sRNA调控接合相关基因的作用机制；④研究HSL处理下候选sRNA的表达变化及其对接合反应的影响，阐明“HSL-SdiA-sRNA”信号通路对接合反应的调节机制，丰富接合反应调控理论，为阻断耐药基因水平转移、发展以QS系统和sRNA为靶点的抗菌新策略提供依据。

细菌耐药如影随形地伴随着抗生素的应用，对人类健康造成巨大威胁。深入研究细菌耐药机制，阻断耐药基因播散具有重大意义。接合（conjugation）是细菌以性菌毛为桥梁进行基因水平转移的过程，是质粒介导耐药基因传播的主要机制。传统观念认为，接合主要由质粒上的接合性元件调控，本课题以调控细菌间相互作用的群体感应系统（quorum sensing，QS）及适应环境变化的sRNA为切入点，利用基因组整合了接合性质粒RP4的大肠埃希菌（E. coli）菌株SM10λπ作为供体菌、具有典型QS系统的铜绿假单胞菌（P. aeruginosa）标准菌株PAO1或不具有QS信号分子的大肠埃希菌菌株EC600为受体菌建立接合模型，采用RNA-seq、Real-time PCR、基因敲除与过表达、报告系统等一系列细胞和分子生物学手段，深入探讨了HSL-SdiA-sRNA信号通路对耐药基因接合转移的调控作用及其具体机制。研究结果发现：1）自然情况下，HSL-SdiA具有抑制SM10λπ-PAO1/EC600接合、限制质粒播散或入侵的功能。在抗生素胁迫下，PAO1 QS系统可表达下调，为细菌通过接合获得耐药基因解除障碍；2）在无特殊环境如抗生素或特定营养元素缺乏情况下，自然培养的SM10λπ中只有极少数sRNA受HSL-SdiA调控，主要有gadY和csrB，两者在敲除sdiA后表达下调，回复sdiA的表达后表达上调；3）过表达gadY能负反馈性地抑制SdiA的表达，从而上调接合关键基因TraI的表达，进而以依赖于HSL存在的方式促进SM10λπ-PAO1/EC600接合。我们的研究结果丰富了耐药基因接合性播散的分子机制，加深了人们对HSL-SdiA-sRNA调控网络及其生物学功能的认识，为抗菌治疗提供了新的策略，具有较高的科学意义和潜在临床价值。