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: 6229434
• 负责人: John C Alverdy
• 金额: $ 24.98万
• 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/6229434
• 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.

严重患者肠道中仅存在铜绿假单胞菌的存在与70％的死亡率有关 - 这是3倍以高于培养这种病原体阴性的匹配的患者。我们建议，在手术压力的宿主的肠道内，物理和化学环境信号会导致铜绿假单胞菌的毒力表型的关键转移。这些影响导致肠道铜绿假单胞菌的行为发生了变化，从而使这种细菌在适当的提示后从懒惰的结肠化剂转移到危及生命的病原体。在此提案中，我们提供了有力的证据表明，铜绿假单胞菌的毒力决定因素，Pa-i凝集素/粘附素，在手术压力的宿主中在致死性肠道脱发中起关键作用。 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紧密连接导致其致命的细胞毒素的细胞细胞转运，3）铜绿假单胞菌的PA-I凝集素通过激活涉及胶囊表达的调节分子的激活来改变上皮紧密的连接渗透性，该分子是胶囊表达的，这是副细胞途径的速率限制密封的速率密封。我们将使用我们已广泛研究的内源性铜绿假单胞菌和培养的肠上皮细胞的新型小鼠模型来检验这些假设。我们的特定目的是：1）确定手术应激（肝切除术）和Cecal注射活孢子虫的铜绿和小鼠的铜绿假单胞菌中PA-I在铜绿假单胞菌中的表达，位置和功能。 2）确定铜绿假单胞菌细胞毒素，外毒素A和弹性蛋白酶的转运途径，响应于纯化的PA-I和精选的生物aeurginosa的突变体，遍及培养的肠上皮细胞（CACO-2）。 3）探索肠上皮屏障功能的PA-I诱导降低的潜在细胞机制。我们建议我们应该重新考虑对脓毒症肠道理论的理解，以包括病原细菌改变其毒力策略的机制，以应对其当地环境中的压力变化。了解病原体用来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疗法，从而避免在更近更接近的地方，从而避免了医院感染。