Gut pathogen virulence and its therapeutic modulation during surgical injury

描述(申请人提供)：脓毒症发生在手术损伤后的病程晚期，称为迟发性脓毒症，现在是创伤、烧伤和择期手术后最常见的死亡原因，并且发病率正在增加。有令人信服的证据表明，促进健康的肠道微生物(即“微生物群”)的丧失以及它被与医疗保健相关的低多样性病原体群落(即“病原体”)所取代，在脓毒症和死亡的免疫病理学中起着重要作用。在过去的3个连续的资助周期中，我们的实验室发现，当肠道病原体受到宿主代偿信号(即阿片类药物、细胞因子、缺血代谢物)在手术损伤期间释放到肠道中时，它们会以一种上下文相关的方式动态表达毒力。在这一建议中，我们假设这些病原体之间的动态毒力表达引导它们表达一种免疫改变的表型，从而导致败血症和死亡。在我们的上一个资金周期中，我们确定了在几种主要病原体的毒力回路中触发生死信号的一个主要因素-磷酸盐(PI)。当磷酸盐丰富时，微生物的磷调节通路凌驾于宿主信号的毒力触发效应之上，而当磷酸盐耗尽时，例如在损伤期间发生在肠道中，病原菌的毒力激活被增强，导致免疫改变、促炎和致死表型的表达。我们合成了一种新的化合物，通过将PI共价键合到具有细胞保护作用的高分子量聚乙二醇上，在损伤期间保持肠道磷酸盐的丰度，这里称为PI-PEG。在这里，我们将测试一种假设，即在手术损伤期间使用PI-PEG保持肠道磷酸盐丰度将维持“微生物群”的健康促进功能，减弱“病原体”的毒力，并防止在肠道接种实际的多病原体群落的小鼠的败血症和死亡，这些实际多病原体群落存在于晚发性败血症患者的危重疾病中。因此，我们将1.探讨PI-PEG介导的抑制迟发性脓毒症患者肠道病原菌毒力表达的机制2.通过肠道上皮细胞(IECS)和树突状细胞(DC)，确定微生物毒力动态表达在脓毒症免疫病理中的作用以及PI-PEG对其调节作用；3.通过检测创伤小鼠暴露于败血症患者的人类病原体群落时，肠道内宿主-病原体相互作用的系统生物学，阐明PI-PEG的保护作用。综上所述，本研究对脓毒症的研究具有重要意义。我们将利用荟萃组学来进一步了解当小鼠被从脓毒症危重患者分离的实际人类病原体群落定居时，动态毒力激活所涉及的系统生物学。由于PI-PEG对微生物的生长没有影响，因此我们可以从机理上了解微生物在手术损伤时在肠道中的行为，它们对免疫系统的影响，以及PI-PEG是如何调节这种相互作用的。

DESCRIPTION (provided by applicant): Sepsis occurring late in the course following surgical injury, termed "late onset sepsis" is now the most common cause of death following trauma, burns, and elective surgery and is increasing in incidence. There is compelling evidence that loss of health promoting intestinal microbes (i.e "microbiome") and its replacement by low diversity communities of healthcare associated pathogens (i.e "pathobiome") plays a major contributing role in the immunopathology of sepsis and mortality. Over the last 3 continuous cycles of funding our laboratory has discovered that intestinal pathogens dynamically express virulence in a context dependent manner when they are "cued" by host compensatory signals (i.e opioids, cytokines, ischemic metabolites) released into the gut during surgical injury. In thi proposal we hypothesize that dynamic virulence expression among such pathogens directs them to express an immune altering phenotype that results in sepsis and mortality. In our last cycle of funding we identified a major factor that triggers a life or death signal within the virulence circuitry of several major pathogens- phosphate (Pi). When phosphate is abundant, microbial phosphoregulatory pathways override the virulence triggering effect of host signals whereas when Pi is depleted, such as occurs in the gut during injury, pathogen virulence activation is enhanced leading to the expression of immune- altering, pro-inflammatory, and lethal phenotypes. We synthesized a novel compound to maintain intestinal phosphate abundance during injury by covalently bonding Pi to a cytoprotective high MW polyethylene glycol, herein termed Pi-PEG. Here we will test the hypothesis that maintaining gut phosphate abundance with Pi-PEG during surgical injury will maintain the health promoting function of the "microbiome", attenuate the virulence of the "pathobiome", and prevent sepsis and mortality in mice intestinally inoculated with the actual multi- pathogen communities that are present in patients with late onset sepsis who are critically ill. Therefore we will 1. Investigate the mechanisms responsible for Pi-PEG-mediated inhibition of microbial virulence expression among virulent and resistant pathogens isolated from the gut of surgical patients with late onset sepsis 2. Define the role of dynamic microbial virulence expression on the immunopathology of sepsis and its modulation by Pi-PEG using intestinal epithelial cells (IECs) and dendritic cells DCs and 3. Elucidate the protective action of Pi-PEG by examining the systems biology of host- pathogen interactions in the gut when injured mice are exposed to human pathogen communities from patients with sepsis. Taken together, the proposed studies are highly significant for the field of sepsis research. We will leverage meta-omics to further our understanding of the systems biology involved in dynamic virulence activation when mice are colonized by actual human pathogen communities isolated from septic critically ill patients. Because Pi-PEG has no effect on microbial growth, we can gain mechanistic insight into how microbes behave in the gut when exposed to surgical injury, their effect on the immune system and how this interaction is modulated by Pi-PEG.