PSEUDOMONAS' EFFECTS ON THE GUT BARRIER FROM SURGERY

外科危重患者肠道中铜绿假单胞菌的存在与70%的死亡率相关-比培养阴性的匹配患者增加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植者转变为威胁生命的病原体。在该提议中，我们提供了强有力的证据，证明铜绿假单胞菌中的毒力决定因子PA-1凝集素/粘附素在手术应激宿主中的致死性肠源性脓毒症中起关键作用。本项目中要检验的假设是：1）铜绿假单胞菌的PA-1凝集素在体内响应于肠道中的环境因素（包括手术应激后的pH、氧化还原状态和去甲肾上腺素）而表达（肝切除术）2）铜绿假单胞菌的PA-1凝集素在细胞间紧密连接水平诱导上皮通透性缺陷，导致其致死性细胞毒素的细胞旁转运，和3）铜绿假单胞菌的PA-1凝集素通过激活参与闭合蛋白（细胞旁途径的限速密封）表达的调节分子来改变上皮紧密连接通透性。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已经广泛研究的培养的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以及在体外操纵其物理微环境（pH、氧化还原、渗透压、去甲肾上腺素）后，从小鼠的不同组织部位收获的铜绿假单胞菌中PA-1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和弹性蛋白酶响应于纯化的PA-1和选定的活铜绿假单胞菌突变体穿过培养的肠上皮细胞（Caco-2）的转运途径。3)探索PA-I诱导肠上皮屏障功能降低的潜在细胞机制。我们建议，我们应该重新思考我们对脓毒症的肠道理论的理解，包括致病菌改变其毒力策略，以应对当地环境的压力变化的机制。了解病原体用于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，这些治疗方法可以在重症患者的护理中更近距离地避免医院感染。

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.