金黄色葡萄球菌SprC下调转录调节子IraC翻译致杀白细胞素lukED转录抑制的分子机制

It was shown by our group that small RNA SprC of Staphylococcus aureus (S. aureus) down-regulates the expression of transcriptional regulator IraC by binding to iraC mRNA, and further inhibits the transcription of leukotoxin ED (lukED) gene. However the mechanisms of this regulatory network are unknown. IraC is a member of AraC family proteins, which are able to regulate genes transcription by directly binding the promoter region of target genes. Previous studies have shown that most of bacterial sRNAs regulate the proteins expression in translation level by binding to the ribosome-binding site (RBS) of the target mRNA; the RNA chaperone Hfq can promote the duplex formation between sRNA and target mRNA, and this binding may trigger the activity of RNase III affecting the stability of target mRNA. Therefore, we speculate that SprC down-regulates the expression of IraC by combining with the RBS of iraC mRNA; Hfq may promote the binding of SprC-iraC mRNA, and RNase III may reduce the stability of target mRNA in complex, which futher leads to the decrease of IraC expression; The down-regulation of the expression of IraC results in the reduction of this protein combining with the lukED, and the regulatory role of IraC on the transcription of lukED will be relieved. In this research project we will elucidate the molecular mechanisms of inhibition of lukED expression by SprC via down-regulation of transcriptional regulator IraC, and explore the roles of Hfq and RNase III in this regulatory network. This study will help us deeply understand the pathogenesis of S. aureus, and give a clue for improving the prevention and therapy strategies of S. aureus infections.

项目组前期研究发现金黄色葡萄球菌小RNA SprC与转录调节子iraC mRNA结合下调其翻译并致杀白细胞素lukED转录抑制，但机制不明。IraC为AraC家族蛋白，具有与靶基因启动子区结合调控转录的特点。细菌sRNA主要通过结合mRNA核糖体结合位点（RBS）影响靶蛋白翻译，RNA伴侣Hfq能促进两者结合，结合后可能触发RNase III活性影响mRNA稳定性。故推测：SprC下调IraC表达可能是通过结合iraC mRNA RBS实现的；Hfq和RNase III可能促进两者结合及降低mRNA的稳定性；IraC表达下降导致其与lukED结合减少，从而解除IraC对lukED的转录调控。本项目拟阐明SprC下调IraC并致lukED转录抑制的分子机制，探讨Hfq及RNase III对该调控网络的影响。研究结果有助于深入了解金黄色葡萄球菌致病机理，为改进该菌感染的预防和治疗策略提供线索。

金黄色葡萄球菌是困扰全球公共卫生及人类健康的重要病原菌，因具有快速获得抗菌药物耐药能力，常常导致抗感染治疗失败，加之引起感染高毒力菌株的增加，严重威胁着人类健康。耐药菌株的持续威胁，迫切需要开发出对细菌演化适应性没有选择压力的新型治疗方法，而干扰毒力因子产生的抗毒力治疗则为一个理想的新选择。细菌非编码小RNA（small non-coding RNA, sRNA）是一类重要的基因表达调节子，可调节细菌的应激适应性及毒力因子表达。本项目研究内容主要为，在金黄色葡萄球菌小RNA SprC与转录调节子AraC家族蛋白IraC mRNA结合下调其翻译并致杀白细胞素LukED转录抑制基础上研究SprC下调lukED转录的分子机制。分子生物学（如同源重组、质粒回补、RACE技术、Northern blot、qRT-PCR、Western blot等）、生物信息学等方法的实验结果显示，SprC通过其 5′-非编码区（5′-UTR）核苷酸与iraC mRNA反义结合形成二聚体，在翻译水平负调控IraC表达。SprC-iraC mRNA 复合物不能触发双链特异性核酸内切酶III（RNase III）的水解活性降低iraC mRNA 稳定性。RNA伴侣Hfq在SprC调控IraC表达调控过程中也不发挥作用。IraC表达下降导致其与lukED结合减少，从而解除IraC对lukED的转录调控。本研究结果探明了SprC在lukED表达调控网络中所起的作用及其分子生物学作用机制，对深入了解金黄色葡萄球菌致病机理、改进金黄色葡萄球菌感染的预防和治疗策略具有十分重要的意义。