绿脓杆菌多重毒力调节子（MvfR）在肠粘膜屏障功能损伤及肠源性脓毒症中的作用研究

Following changes of environmental signals present in the gut during stress, opportunistic bacteria within the gastrointestinal tract, such as the gram-negative bacterium Pseudomonas aeruginosa, may pose a phenotypic change to pathogenic state. Opportunistic Pseudomonas aeruginosa may play a role in the development of bacterial translocation and gut-derived sepsis during stress. Multiple virulence factor regulator (MvfR) modulates the expression of multiple quorum sensing (QS)-regulated virulent states. In this study, we further assume that P. aeruginosa is activated to induce PA-IL production in respond to the opioid peptide morphine through MvfR, disrupting the intestinal epithelial barrier and triggering gut-derived sepsis..We investigated the effects of morphine on intestinal epithelial barrier dysfunction by using P. aeruginosa strain PAO1 as well as corresponding mvfR mutant and complemented strain. P. aeruginosa mvfR mutant was constructed by using CRISPR/Cas9, and then the complemented strain was constructed. TSB with morphine and without morphine was used, and then the experiment of bacterial motility was taken. Virulence expression in P. aeruginosa was examined in vitro by assessing expression of the PA-IL, a barrier disrupting protein. Additionally, mice were implanted with a slow release morphine or placebo pellet with and without intestinal inoculation of P. aeruginosa created by direct cecal injection. Levels of plasma cytokines, bacterial translocation to distant organs were determined 36h later. The demonstration of such effect would suggest a new mechanism for the development of gut-derived sepsis. Our results are helpful for preventing and combating gut-derived sepsis.

在手术、创伤等应激状态下，神经内分泌的改变会引起肠道内条件致病菌绿脓杆菌发生表型变化成为侵袭力强的绿脓杆菌，继而发生细菌移位，导致肠源性脓毒症。研究发现，绿脓杆菌多重毒力调节子（MvfR）可通过细菌密度感应系统识别外部信号分子，如阿片类物质，造成绿脓杆菌表型变化。本项目进一步提出，绿脓杆菌MvfR可通过与吗啡结合，促进PA-IL生成，引起肠粘膜屏障功能损伤，从而触发肠源性脓毒症。.本研究采用CRISPR/Cas9系统及基因互补技术构建绿脓杆菌mvfR基因缺失突变菌株及基因互补菌株，通过体内外实验，进行绿脓杆菌的mvfR基因功能研究；从分子和细胞水平探讨绿脓杆菌MvfR及吗啡对绿脓杆菌PA-IL生成的作用；通过建立吗啡慢性释放的动物模型，探讨绿脓杆菌MvfR在肠粘膜屏障功能损伤及肠源性脓毒症中的作用；揭示肠源性脓毒症发生发展新机制，发现防治绿脓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。在手术、创伤等应激状态下，神经内分泌的改变会引起肠道内条件致病菌绿脓杆菌发生表型变化成为侵袭力强的绿脓杆菌，对肠粘膜屏障进行强有力的破坏，导致肠源性脓毒症。PA-IL是绿脓杆菌分泌的一种能破坏肠粘膜屏障的细胞毒素。绿脓杆菌多重毒力调节子（MvfR）可以通过细菌密度感应系统识别外部信号分子阿片类物质强啡肽，这在绿脓杆菌表型变化（即从非致病性到致病性，从侵袭力弱到侵袭力强）中起重要调节作用，在绿脓杆菌PA-IL的生成以及肠粘膜屏障功能损伤中起重要作用。本研究构建绿脓杆菌mvfR基因缺失菌株及基因互补菌株，通过体内、外实验，进行绿脓杆菌的mvfR基因功能研究；从分子和细胞水平探讨绿脓杆菌MvfR及阿片类物质对绿脓杆菌lecA表达的作用；通过建立动物模型，探讨绿脓杆菌MvfR及阿片类物质在肠粘膜屏障功能损伤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。.研究结果提示：①应用CRISPR/Cas9技术构建绿脓杆菌基因突变菌株仍有困难，可考虑利用转座子技术，并由国外实验室惠赠得到了绿脓杆菌mvfR阴性突变菌株；构建了绿脓杆菌mvfR互补菌株。②绿脓杆菌MvfR对细菌的生长没有影响；阿片受体激动剂（不论是μ型阿片受体激动剂吗啡，还是κ型阿片受体激动剂U50488和地佐辛）对绿脓杆菌的生长也没有影响。③κ受体激动剂（U50488和地佐辛）能增强绿脓杆菌的运动能力，而且该作用是通过MvfR介导的；而μ受体激动剂吗啡则无此作用。④κ受体激动剂地佐辛和U-50488增强绿脓杆菌lecA基因的表达以及绿脓菌素的生成，而且该作用是通过MvfR介导的；μ受体激动剂吗啡则没有该作用。⑤PAO1+地佐辛组小鼠的肠紧密连接蛋白Claudin-1表达较之PAO1组明显降低，而ΔmvfR+地佐辛组与ΔmvfR组相比，Claudin-1的表达变化并不明显。⑥阿片受体激动剂地佐辛可直接作用于肠道内定植的机会性致病菌绿脓杆菌，引起绿脓杆菌表型改变，毒力因子生成增多，加重PA-IL介导的肠粘膜屏障功能损伤，最终引起肠源性脓毒症。