The role of microbial second messenger synthesis in intestinal homeostasis

项目摘要 肠道微生物区系与STING和MyD88途径相互作用以增强健康的粘液分泌和先天 肠道中的免疫激活。粘液调节失调导致微生物诱导的炎症，这是 胃肠道病理，但支持这一过程的机制知之甚少。如果我们能更好地理解 在分子水平上研究宿主粘液与细菌生理的关系，可以设计治疗方案 减轻粘液调节失调引起的炎症。细菌第二信使c-di-GMP(CDG)， 它促进细菌聚集以响应环境提示，刺激脊椎动物的天然免疫 刺痛信号。斑马鱼幼体，Danio rerio，是探索宿主微生物方面的强大模型 相互作用在人类中是保守的。我最近的研究表明斑马鱼的互助性气单胞菌 ZOR0001(A01)分泌人类相关微生物区系中保守的有益因子，增加CDG 粘液反应水平，导致粘液结合粘附素上调，以及A01聚集 斑马鱼幼体肠道粘液丰富的中肠。各种细菌在接触粘液时会增加CDG， 这表明这一概念在细菌中是一种保守的适应策略。我们假设粘液介导的A01 聚集促进健康的粘液分泌，设定适当的炎症基调以防止过度 发炎。与这一假设一致，我在斑马鱼的遗传背景中观察到了高度运动性的A01 粘液分泌细胞减少。此外，我发现治疗斑马鱼突变体容易自发 产生高水平CDG的A01超聚集性突变体可以缓解肠道炎症 肠内中性粒细胞流入。因此，我们建议表征A01 CDG信号的分子基础。 并确定正常的CDG-STING信号是否有助于肠道内稳态。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)研究如何刺痛 A01产生的CDG的感应在体内调节宿主炎症和肠道粘液稳态。研究 设计：使用我的A01 CDG报告菌株，我将首先筛选与粘液介导的CDG有关的A01基因 这些突变体的体外合成及其与野生型肠道CDG信号和分布模式的比较 斑马鱼肠道中的A01。为了了解CDG调节在互惠互利和宿主肠道健康中的作用， 粘液感应缺陷的A01突变体将被单关联到转基因斑马鱼幼体中，从而使 肠道粘液分泌细胞和炎症反应的量化。我也会产生不足的刺痛 斑马鱼不能感知CDG，通过表型特征和单细胞RNAseq比较， 与野生型和MyD88斑马鱼相比，它们对A01和细菌产物(如CDG和LPS)的反应。 这项研究将提高我们对生物和医学上重要过程的理解 宿主与微生物的相互作用。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.