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 this 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.
We have discovered that during surgical injury, intestinal microbes dynamically express virulence in a context dependent manner by sensing and responding to host- derived physiologic cues that can lead to immune activation and a lethal phenotype. In our recent funding cycle we discovered that the local concentration of phosphate (Pi) is a universal cue among hospital acquired pathogens that enhances microbial virulence expression when Pi is deficient yet inhibits virulence when Pi is abundant. Therefore as a countermeasure to prevent intestinal microbial virulence activation and its consequences during surgical injury, we have synthesized a unique phosphate based polymer (Pi-PEG) that inhibits virulence activation in intestinal pathogens and protects against mortality and now seek to determine its mechanism of action and applicability in relevant cellular and animal models of surgical injury.
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