PSEUDOMONAS' EFFECTS ON THE GUT BARRIER FROM SURGERY

手术对假单胞菌对肠道屏障的影响

基本信息

批准号：
6608389
负责人：
John C Alverdy
金额：
$ 1万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-02-01 至 2005-01-31
项目状态：
已结题

项目摘要

The mere presence of Pseudomonas aeruginosa in the intestine of critically ill surgical patients is associated with a 70% mortality rate--a 3 fold increase above matched patients who culture negative for this pathogen. We propose that within the intestinal tract of a surgically stressed host, physical and chemical environmental signals cause critical shifts in the virulence phenotype of P. aeruginosa. These effects result in a change in the behavior of intestinal P. aeruginosa, causing this bacteria, upon proper cue, to shift from an indolent colonizer to a life-threatening pathogen. In this proposal we provide strong evidence that a virulence determinant in P. aeruginosa, the PA-I lectin/adhesin, plays a key role in lethal gut- derived sepsis in a surgically stressed host. The hypotheses to be tested in this project are: 1) the PA-I lectin of P. aeruginosa is expressed in vivo in response to environmental cues in the intestinal tract including pH, redox state, and norepinephrine following surgical stress (hepatectomy) 2) the PA-I lectin of P.aeruginosa induces an epithelial permeability defect at the level of the intercellular tight junction resulting in paracellular transport of its lethal cytotoxins, and 3) the PA-I lectin of P. aeruginosa alters epithelial tight junctional permeability by activation of regulatory molecules involved in the expression of occludin, the rate limiting seal of the paracellular pathway. We will test these hypotheses using a novel mouse model of endogenous P. aeruginosa sepsis and cultured intestinal epithelial cells that we have extensively studied. Our specific aims to test these hypotheses are: 1) Determine the expression, location, and function of PA-I in P. aeruginosa harvested from different tissue sites in mice following surgical stress (hepatectomy) and cecal injection of live P. aeruginosa and following in vitro manipulation of the its physical microenvironment (pH, redox, osmolality, norepinephrine). 2) Determine the route of transport of the P. aeruginosa cytotoxins, exotoxin A and elastase, across cultured intestinal epithelial cells (Caco-2) in response to purified PA-I and selected mutants of live P. aeurginosa. 3) Explore potential cellular mechanisms of PA-I-induced decreases in intestinal epithelial barrier function. We propose that we should rethink our understanding of the gut theory of sepsis to include mechanisms by which pathogenic bacteria alter their virulence strategies in response to stressful changes in their local environment. Understanding the virulence determinants and cellular mechanisms that pathogens use to adhere to and modify the intestinal epithelial barrier may lead to therapies which can avoid nosocomial infection at a more proximate point in the care of the critically ill.

危重手术患者肠道中仅存在铜绿假单胞菌就导致 70% 的死亡率，比该病原体培养阴性的匹配患者死亡率增加了 3 倍。我们认为，在手术应激宿主的肠道内，物理和化学环境信号导致铜绿假单胞菌毒力表型的关键转变。这些影响导致肠道铜绿假单胞菌的行为发生变化，导致这种细菌在适当的提示下从惰性定殖者转变为危及生命的病原体。在本提案中，我们提供了强有力的证据，证明铜绿假单胞菌的毒力决定因子 PA-I 凝集素/粘附素在手术应激宿主的致命性肠源性败血症中发挥着关键作用。本项目要测试的假设是：1) 铜绿假单胞菌的 PA-I 凝集素在体内表达，以响应肠道环境提示，包括手术应激（肝切除术）后的 pH、氧化还原状态和去甲肾上腺素 2)铜绿假单胞菌的 PA-I 凝集素在细胞间紧密连接水平诱导上皮细胞通透性缺陷，导致其致命细胞毒素的旁细胞转运，并且3)铜绿假单胞菌的PA-I凝集素通过激活参与occludin表达的调节分子来改变上皮紧密连接通透性，occludin是细胞旁通路的限速密封。我们将使用内源性铜绿假单胞菌败血症的新型小鼠模型和我们广泛研究的培养肠上皮细胞来测试这些假设。我们测试这些假设的具体目的是：1) 确定在手术应激（肝切除术）和盲肠注射活铜绿假单胞菌后从小鼠不同组织部位收获的铜绿假单胞菌中 PA-I 的表达、位置和功能体外操纵其物理微环境（pH、氧化还原、渗透压、去甲肾上腺素）。 2)确定铜绿假单胞菌细胞毒素、外毒素A和弹性蛋白酶响应纯化的PA-I和活铜绿假单胞菌的选定突变体跨培养的肠上皮细胞(Caco-2)的转运途径。 3) 探索PA-I诱导肠上皮屏障功能下降的潜在细胞机制。我们建议我们应该重新思考我们对脓毒症肠道理论的理解，包括致病菌改变其毒力策略以应对当地环境压力变化的机制。了解病原体用于粘附和改变肠上皮屏障的毒力决定因素和细胞机制可能会导致在危重病人的护理中更近距离地避免院内感染的治疗方法。