PSEUDOMONAS' EFFECTS ON THE GUT BARRIER FROM SURGERY

外科危重患者肠道中仅铜绿假单胞菌的存在就与70%的死亡率有关--比培养出这种病原体阴性的匹配患者高出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变化。这些影响导致肠道铜绿假单胞菌的行为发生变化，使这种细菌在适当的提示下，从一个懒惰的定殖者转变为威胁生命的病原体。在这项建议中，我们提供了强有力的证据表明，铜绿假单胞菌中的一个毒力决定因素，PA-I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，以响应肠道环境因素，包括手术应激(肝切除)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凝集素诱导细胞间紧密连接水平的上皮通透性缺陷，从而导致其致命细胞毒素的旁细胞运输，以及3)铜绿假单胞菌的PA-I凝集素通过激活参与阻滞素表达的调节分子来改变上皮紧密连接的通透性。我们将使用一种新的内源性铜绿假单胞菌败血症小鼠模型和我们广泛研究的培养的肠道上皮细胞来验证这些假设。我们的具体目的是验证这些假设：1)确定PA-I在手术应激(肝切除)和盲肠注射活的铜绿假单胞菌以及体外操作其物理微环境(pH、氧化还原、渗透压、去甲肾上腺素)后，在小鼠不同组织部位的表达、定位和功能。2)确定铜绿假单胞菌细胞毒素，外毒素A和弹性蛋白酶，通过培养的肠上皮细胞(Caco-2)对纯化的PA-I和选定的活体铜绿假单胞菌突变体的转运途径。3)探讨PA-I降低肠上皮屏障功能的可能细胞机制。我们建议我们应该重新思考我们对脓毒症的肠道理论的理解，以包括病原菌改变其毒力策略以应对其局部环境的应激变化的机制。了解病原体用来黏附和修改肠道上皮屏障的毒力决定因素和细胞机制，可能会导致在治疗危重病人的过程中更接近避免医院感染的治疗方法。

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.