PSEUDOMONAS' EFFECTS ON THE GUT BARRIER FROM SURGERY

手术对假单胞菌对肠道屏障的影响

• 批准号: 6570142
• 负责人: John C Alverdy
• 金额: $ 3.34万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/6570142
• 关键词: Pseudomonas aeruginosa; adhesin; bacteria infection mechanism; bacterial genetics; bacterial proteins; bacterial toxins; biological signal transduction; elastases; exotoxins; gastrointestinal epithelium; gene expression; hepatectomy; laboratory mouse; lectin; membrane permeability; mutant; myosin light chain kinase; northern blottings; phosphorylation; physiologic stressor; postoperative complications; protein structure function; tight junctions; tissue /cell culture; western blottings

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.

仅在危重外科患者肠道中存在铜绿假单胞菌就与70%的死亡率相关，比该病原体培养阴性的匹配患者增加了3倍。我们提出，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变化。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一个懒惰的定植者转变为一个危及生命的病原体。在这项提议中，我们提供了强有力的证据，证明P. aeruginosa的毒力决定因素pa - 1凝集素/粘附素在手术应激宿主的致死性肠源性败血症中起关键作用。本项目要检验的假设有：1) P.aeruginosa的pa - 1凝集素在体内表达，以响应手术应激（肝切除术）后肠道内的环境信号，包括pH、氧化还原状态和去甲肾上腺素。2)P.aeruginosa的pa - 1凝集素在细胞间紧密连接水平诱导上皮通透性缺陷，导致其致命细胞毒素的细胞旁运输。3) P. aeruginosa的PA-I凝集素通过激活参与occludin表达的调控分子来改变上皮紧密连接的通透性，occludin是细胞旁通路的限速密封。我们将使用一种新的内源性铜绿假单胞菌脓毒症小鼠模型和我们广泛研究的培养肠上皮细胞来验证这些假设。我们验证这些假设的具体目的是：1)确定pa - 1在手术应激（肝切除术）和活体P. aeruginosa盲肠注射后小鼠不同组织部位收集的P. aeruginosa中的表达、位置和功能，并对其物理微环境（pH、氧化还原、渗透压、去甲肾上腺素）进行体外处理。2)确定铜绿假单胞菌细胞毒素、外毒素A和弹性蛋白酶在体外培养肠上皮细胞（Caco-2）中的转运途径，以响应纯化的pa - 1和选择的活的铜绿假单胞菌突变体。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，包括致病细菌在应对当地环境压力变化时改变其毒力策略的机制。了解病原体粘附和修饰肠上皮屏障的毒力决定因素和细胞机制，可能会导致治疗方法的出现，从而在更接近危重病人的护理中避免医院感染。