Pseudomonas' effects on the gut barrier from surgery

描述（由申请人提供）：在过去的10年中，我们的实验室在了解手术损伤后肠道定植的医院内病原体如何导致致命败血症方面取得了重大进展。我们发现，铜绿假单胞菌（一种定植于多达50%的危重患者的肠道的人类机会性病原体）直接响应于在手术损伤期间释放到肠道中的宿主应激化合物，将其表型从惰性定植者转变为致死病原体，所述应激化合物包括：1.）免疫元件（干扰素3），2.）阿片类（吗啡、强啡肽），和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，铜绿假单胞菌定植的肠道部位也会发生磷酸盐耗竭，并通过与群体感应系统（铜绿假单胞菌和其他细菌中毒力基因激活的主要机制）交叉的高度敏感的磷酸盐调节机制增强铜绿假单胞菌对宿主应激化合物的反应性。我们还发现，在富含磷酸盐的环境中，铜绿假单胞菌毒力表达在宿主应激化合物的存在下显著减弱，并且其在小鼠肠中的致死作用在手术损伤期间完全消除。我们现在建议实现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）。确定磷酸盐消耗和宿主组织BSC协同作用以在手术损伤后将铜绿假单胞菌转变为肠中的致死轨迹的分子机制2.）确定手术损伤后体内肠道中铜绿假单胞菌毒力激活所涉及的位点特异性分子机制。3.）第三章设计富含磷酸盐的合成粘蛋白，其在手术损伤期间存在于肠道中时使铜绿假单胞菌分子沉默。4.）使用定制的PCR阵列确定危重病人的肠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。该提案的结果将为致死性肠源性脓毒症的发病机制提供前所未有的分子细节水平，并为发现和测试干扰致命病原体毒力激活的化合物提供新的治疗方向，这些病原体定植在危重患者的肠道中。 公共卫生相关性：该提案旨在了解铜绿假单胞菌（一种常见的病原体，定植于手术损伤患者的肠道）的分子基础，由于手术损伤期间肠道中存在的局部环境线索，铜绿假单胞菌将其表型从惰性定植者转变为致命病原体。使用线虫，小鼠和人类肠内容物从严重受伤的患者，我们将阐明铜绿假单胞菌表达对肠上皮细胞的致死表型的分子基础，并确定新的治疗靶点和非抗生素化合物，分子沉默这种病原体表达致死表型。

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.