Pseudomonas' effects on the gut barrier from surgery

描述（由申请人提供）：在过去的10年里，我们的实验室在了解手术损伤后肠道定植的医院病原体如何导致致死性败血症方面取得了重大进展。我们发现铜绿假单胞菌（Pseudomonas aeruginosa）是一种人类机会性病原体，在多达50%的危重患者的肠道中定植，它的表型从惰性定植者转变为致命病原体，直接响应宿主在手术损伤期间释放到肠道的应激化合物，包括：1.)免疫成分（干扰素3），2.)阿片类物质（吗啡，肌啡），3.)细胞缺氧的最终产物（腺苷）。最近，我们证明了在手术损伤期间，P. aeruginosa定植的肠道部位也发生了磷酸盐消耗，并通过与群体感应系统交叉的高度敏感的磷酸盐调节机制增强了P. aeruginosa对宿主应激化合物的反应性，群体感应系统是P. aeruginosa和其他细菌中毒力基因激活的主要机制。我们还发现，在富含磷酸盐的环境中，P. aeruginosa毒力表达在宿主应激化合物的存在下显着减弱，其在小鼠肠道中的致死作用在手术损伤期间完全消除。我们现在建议完成4个目标，将我们正在进行的工作扩展到其自然转化和治疗终点，包括：1.确定磷酸盐耗损和宿主组织BSCs协同作用将P. aeruginosa在手术损伤后肠内转移到致死轨迹的分子机制2.确定手术损伤后P. aeruginosa在体内肠道内毒力激活的位点特异性分子机制。3)设计富磷酸盐的合成粘蛋白，在手术损伤时在肠道中抑制铜绿假单胞菌。4.)利用定制PCR阵列确定危重病人肠道内容物将铜绿假单胞菌的分子特征转变为致命轨迹的机制。这一建议的结果将提供致死性肠源性败血症发病机制的前所未有的分子细节水平，并为发现和测试干扰在危重患者肠道定植的致死性病原体的毒力激活的化合物提供新的治疗方向。公共卫生相关性：本研究旨在了解铜绿假单胞菌（一种定殖于手术损伤患者肠道的常见病原体）在手术损伤期间肠道局部环境提示下将其表型从惰性定植者转变为致命病原体的分子基础。利用线虫、小鼠和来自重伤患者的人类肠道内容物，我们将阐明铜绿假单胞菌对肠上皮表达致死性表型的分子基础，并确定新的治疗靶点和非抗生素化合物，使这种病原体在分子上表达致死性表型。

DESCRIPTION (provided by applicant): Over the past 10 years, our laboratory has made major advances in understanding how nosocomial pathogens that colonize the gut following surgical injury cause lethal sepsis. We discovered that Pseudomonas aeruginosa, a human opportunistic pathogen that colonizes the gut of as many as 50% of critically ill patients, shift its phenotype from that of indolent colonizer to lethal pathogen in direct response to host stress compounds released into the gut during surgical injury that include: 1.) immune elements (interferon 3), 2.) opioids (morphine, dynorphin), and 3.) end-products of cellular hypoxia (adenosine). Most recently we demonstrated that phosphate depletion also develops at intestinal sites of P. aeruginosa colonization during surgical injury and enhances the responsiveness of P. aeruginosa to host stress compounds via highly sensitive phosphate regulatory mechanisms that intersect with the quorum sensing system, the major mechanism of virulence gene activation in P. aeruginosa and other bacteria. We also discovered that in a phosphate rich environment, P. aeruginosa virulence expression is markedly attenuated in the presence of host stress compounds and its lethal effect in the mouse intestine completely abrogated during surgical injury. We now propose to accomplish 4 aims to extend our ongoing work to its natural translational and therapeutic endpoint that include: 1.) determining the molecular mechanisms by which phosphate depletion and host-tissue BSCs synergize to shift P. aeruginosa into a lethal trajectory in the intestine following surgical injury 2. ) determining the site-specific molecular mechanisms involved in P. aeruginosa virulence activation in the intestinal tract in vivo following surgical injury. 3.) designing phosphate rich synthetic mucins that molecularly silence P. aeruginosa when present in the intestinal tract during surgical injury. 4.) determining the mechanisms by which intestinal contents from critically ill humans shift the molecular signature of P. aeruginosa into a lethal trajectory using custom PCR arrays. Results from this proposal will provide an unprecedented level of molecular detail on the pathogenesis of lethal gut-derived sepsis and provide novel therapeutic direction for the discovery and testing of compounds that interfere with virulence activation of lethal pathogens that colonize the gut of critically ill patients. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand the molecular basis by which Pseudomonas aeruginosa, a common pathogen colonizing the gut of surgically injured patients, shifts its phenotype from indolent colonizer to lethal pathogen as a result of local environmental cues present in the intestine during surgical injury. Using nematodes, mice, and human intestinal contents from critically injured patients, we will elucidate the molecular basis by which P. aeruginosa expresses a lethal phenotype against the intestinal epithelium and identify novel therapeutic targets and non-antibiotic compounds that molecularly silence this pathogen from expressing a lethal phenotype.