大肠杆菌密度感应调节子C(QseC)在肠道细菌移位及肠源性脓毒症中的作用研究

Following changes of environmental signals present in the gut that accompanies trauma, commensal bacteria within the gastrointestinal tract, such as the gram-negative bacterium Escherichia coli, may pose a phenotypic change to pathogenic state. Commensal bacteria may play a role in the development of bacterial translocation and gut origin sepsis following trauma. Regulation of the bacterial transition to all forms involves the quorum sensing system. The quorum sensing Escherichia coli regulator C(QseC) is the histidine sensor kinase that detect autoinducer-3/norepinephrine. However, the role of QseC in the gut origin sepsis caused by Escherichia coli remains unclear..We investigated the effects of norepinephrine on bacterial translocation by using the commensal E.coli strain as well as corresponding qseC mutant and complemented strain. Non-pathogenic human commensal E.coli strains used in this study were obtained from the feces of healthy individuals. Then E.coli qseC mutant and the complemented strain were constructed. LB with norepinephrine and without norepinephrine was used, and then the experiment of bacterial motility was taken. Additionally, the selective noradrenergic neurotoxic agent 6-hydroxydopamine(6-OHDA) was employed in a rat model of trauma-induced norepinephrine release, and rats were treated with three E.coli strains respectively. Levels of plasma endotoxin, bacterial translocation to distant organs were determined 24h later. The demonstration of such effect would suggest a new mechanism for the development of gut origin sepsis. Our results are helpful for preventing and combating gut origin sepsis.

在创伤等应激状态下，胃肠道神经内分泌的改变会引起肠道内非致病性共生大肠杆菌发生表型变化，继而发生细菌移位，导致肠源性脓毒症。大肠杆菌密度感应调节子C（QseC）能识别外部信号分子（如去甲肾上腺素），在细菌表型变化的密度感应调节中起重要作用。然而，在肠道共生大肠杆菌发生细菌移位引发肠源性脓毒症的过程中，大肠杆菌QseC的作用如何？目前少有相关报道。.本研究从健康成人粪便中分离得到非致病性共生大肠杆菌，采用Red同源重组及基因互补技术构建大肠杆菌qseC基因缺失突变菌株及基因互补菌株，从分子和细胞水平探讨大肠杆菌QseC及去甲肾上腺素对细菌运动能力的作用；通过建立去甲肾上腺素一过性大量释放的动物模型，探讨大肠杆菌QseC在肠道细菌移位及肠源性脓毒症中的作用；揭示肠源性脓毒症发生发展的新机制，发现防治大肠杆菌引起的肠源性脓毒症可能作用的分子药物靶标。

脓毒症是严重创伤、烧伤、休克、大手术后常见的并发症，也是外科危重患者主要的死亡原因之一。在创伤等应激状态下，胃肠道神经内分泌的改变会引起肠道内非致病性大肠杆菌发生表型变化，继而发生细菌移位，导致肠源性脓毒症。大肠杆菌密度感应调节子C（Quorum sensing E. coli regulator C, QseC）可以识别外部信号分子（如自诱导物3（AI-3）、去甲肾上腺素（NE）等）并与之结合，这在细菌表型变化的密度感应调节中起重要作用。本研究采用Red 同源重组技术及基因互补技术构建大肠杆菌qseC基因缺失突变菌株及基因互补菌株，通过体内、外实验，进行大肠杆菌的qseC基因功能研究；从分子和细胞水平探讨大肠杆菌QseC及去甲肾上腺素对大肠杆菌运动能力的作用；通过建立去甲肾上腺素一过性大量释放的动物模型，探讨大肠杆菌QseC在肠道细菌移位及肠源性脓毒症中的作用；揭示肠源性脓毒症发生发展的新机制，发现防治大肠杆菌引起的肠源性脓毒症可能作用的分子药物靶标，为防治肠源性脓毒症等后续研究提供实验依据。. 研究结果提示：①应用生化鉴定和16S rDNA全序列测定，可以快速准确地鉴定大肠杆菌；②大肠杆菌QseC对细菌的生长没有影响；③大肠杆菌QseC缺失后其运动能力显著下降，而QseC互补菌株又部分恢复了其运动能力，表明QseC对大肠杆菌的运动具有重要调控作用；④去甲肾上腺素对大肠杆菌运动能力有直接促进作用，且该作用是通过QseC介导的；⑤大肠杆菌QseC受体缺陷，去甲肾上腺素-QseC信号链中断，细菌运动能力减弱，最终导致细菌移位发生率下降，说明阻断该通路可抑制肠道细菌移位，QseC-去甲肾上腺素通路在肠道大肠杆菌移位以及肠源性脓毒症中起重要作用。