The role of microbial second messenger synthesis in intestinal homeostasis

项目摘要 肠道微生物群与STING和Myd 88通路相互作用，以加强健康的粘液分泌和先天性 肠道内的免疫激活粘液失调会导致微生物诱导的炎症， 胃肠道病变，但这一进程的机制基础知之甚少。如果我们更好地理解 宿主粘液和细菌生理之间的关系在分子水平上，我们可以设计治疗方法， 以减轻由粘液失调引起的炎症。细菌第二信使c-di-GMP（cdG）， 促进细菌聚集以响应环境信号，刺激脊椎动物先天免疫 STING信号。斑马鱼幼体是探索宿主微生物方面的一个强有力的模型 在人类中保守的相互作用。我最近的研究表明一种斑马鱼互利共生的韦氏气单胞菌 ZOR 0001（A01），其分泌在人类相关微生物群中保守的有益因子，增加cdG 水平，导致粘液结合粘附素的上调，以及A01在细胞中的聚集。 幼斑马鱼肠中富含粘液的中肠。当暴露于粘液时，各种细菌增加cdG， 表明这一概念是细菌中保守的适应性策略。我们假设粘液介导的A01 聚集促进健康的粘液分泌，设置适当的炎症基调，以防止过量 炎症与这一假设相一致，我观察到在斑马鱼遗传背景中具有高度能动性的A01， 粘液分泌细胞减少。此外，我发现，治疗一个斑马鱼突变体， 具有产生高水平cdG的A01超聚集突变体的肠道炎症可以减轻 肠中性粒细胞内流。因此，我们建议表征A01 cdG信号转导的分子基础， 宿主并确定正常的cdG-STING信号传导是否有助于肠内稳态。具体目标：（1） 确定宿主粘液促进A01 cdG合成的遗传基础，以及（2）研究STING如何 A01产生的cdG的感应在体内调节宿主炎症和肠粘液稳态。研究 设计：利用我的A01 cdG报告菌株，我将首先筛选参与粘液介导的cdG的A01基因 体外合成并比较这些突变体与野生型肠cdG信号传导和分布模式 斑马鱼肠道中的A01。为了了解cdG调节在互利共生和宿主肠道健康中的作用， A01粘液敏感缺陷突变体将被单结合到转基因斑马鱼幼虫中， 肠粘液分泌细胞和炎症反应的定量。我也会产生刺痛缺陷 斑马鱼不能感知cdG，通过表型表征和单细胞RNAseq， 相对于野生型和myd 88斑马鱼，它们如何响应A01和细菌产物如cdG和LPS。 这项研究将提高我们对生物学和医学重要过程的理解，这些过程支撑着 宿主-微生物相互作用由于炎症性胃肠道疾病的发病率正在上升，我们的长期目标是 我们的工作是将我们的发现转化为促进人类肠道健康的策略。

Project Summary Enteric microbiota interact with STING and Myd88 pathways to reinforce healthy mucus secretion and innate immune activation in the intestine. Mucus dysregulation precipitates microbe-induced inflammation, a driver of GI pathologies, but the mechanisms underpinning this process are poorly understood. If we better understood the relationship between host mucus and bacterial physiology at the molecular level, we could design therapies to alleviate inflammation that arises from mucus dysregulation. The bacterial second messenger c-di-GMP (cdG), which promotes bacterial aggregation in response to environmental cues, stimulates vertebrate innate immune STING signaling. The larval zebrafish, Danio rerio, is a powerful model for exploring aspects of host-microbe interactions conserved in humans. My recent studies indicate a zebrafish mutualist Aeromonas veronii ZOR0001(A01), which secretes beneficial factors conserved in human-associated microbiota, increases cdG levels in response to mucus, leading to upregulation of a mucus-binding adhesin, and A01 aggregation in the mucus-rich mid gut of the larval zebrafish intestine. Various bacteria increase cdG when exposed to mucus, indicating this concept is a conserved adaptive strategy in bacteria. We hypothesize that mucus-mediated A01 aggregation promotes healthy mucus secretion, setting a proper inflammatory tone to prevent excess inflammation. Consistent with this hypothesis I observe highly motile A01 in a zebrafish genetic background with decreased mucus-secreting cells. Furthermore, I found that treating a zebrafish mutant prone to spontaneous intestinal inflammation with a A01 hyper-aggregating mutant that produces high levels of cdG can alleviate intestinal neutrophil influx. Therefore, we propose to characterize the molecular basis of A01 cdG signaling in the host and determine if normal cdG-STING signaling contributes to intestinal homeostasis. Specific Aims: (1) determine the genetic basis by which host mucus prompts A01 cdG synthesis, and (2) investigate how STING sensing of A01-produced cdG modulates host inflammation and intestinal mucus homeostasis in vivo. Research Design: Using my A01 cdG reporter strain, I will first screen for A01 genes involved in mucus-mediated cdG synthesis in vitro and compare intestinal cdG signaling and distribution patterns of these mutants to wild type A01 in the zebrafish intestine. To understand the role of cdG regulation in mutualism and host intestinal health, A01 mutants deficient in mucus-sensing will be monoassociated into transgenic larval zebrafish that allow quantification of intestinal mucus-secreting cells and inflammation responses. I will also generate sting deficient zebrafish incapable of sensing cdG and compare, through phenotypic characterization and single cell RNAseq, how they respond to A01 and bacterial products such as cdG and LPS relative to wild type and myd88 zebrafish. This research will improve our understanding of biologically and medically important processes that underpin host-microbe interactions. Because rates of inflammatory GI disorders are on the rise, the long-term goal of our work is to translate our findings into strategies for promoting intestinal health in humans.