Exploring the Ecological Role of Rothia mucilaginosa and It's Iron Binding Siderophore Enterobactin

探索 Rothia mucilaginosa 及其铁结合铁载体肠杆菌素的生态作用

基本信息

批准号：
10366025
负责人：
Marcelo Freire
金额：
$ 29.25万
依托单位：
J. CRAIG VENTER INSTITUTE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-03-05 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10366025
关键词：
3-Dimensional Actinobacteria class Address Affinity Anti-Inflammatory Agents Back Bacteria Binding Biochemical Process Biological Assay Biological Models Body Fluids Carrier Proteins Catechols Cell Line Cell Separation Cells Chelating Agents Coculture Techniques Communities Complex Confocal Microscopy Dental Plaque Dose Ecology Enterobactin Environment Enzymes Epithelial Cells Escherichia coli Exposure to Ferritin Flow Cytometry Fluorescence-Activated Cell Sorting Foundations Free Radicals Future Gene Cluster Genes Genetic Transcription Genome Goals Growth Gut Mucosa Human Human Microbiome Human body Immune system In Vitro Incubated Individual Interdisciplinary Study Iron Iron Chelating Agents Knowledge Label Life Light Measurement Measures Metabolic Microbial Biofilms Microscopy Molecular Monitor Mucous Membrane Oral Oral cavity Oral health Oral mucous membrane structure Organism Outcome Oxidative Stress Pathogenicity Phenotype Play Population Production Proteins Publishing Reactive Oxygen Species Reporter Reporter Genes Research Rhodamine Role Saliva Sentinel Siderophores Signal Transduction Streptococcus mutans Streptococcus salivarius System Taxonomy Testing Tissues Transferrin Work bacterial community commensal bacteria comparative cytokine experimental study extracellular gut microbiota host microbiota host-microbe interactions human model in vivo interest iron metabolism member metagenomic sequencing metatranscriptomics microbial microbial host microbiome research multiple omics nasal microbiota oral bacteria oral cavity epithelium oral commensal oral microbial community receptor response rho small molecule three-dimensional modeling transcriptomics

项目摘要

Project Summary The oral cavity is a highly diverse microbial environment, consisting of >2000 bacterial, archaeal, and fungal species most of which have not been functionally characterized. Numerous studies have identified that oral Rothia mucilaginosa, a Gram-positive oral commensal Actinobacteria, is highly abundant in saliva and dental plaque in global human populations, however, its ecological role is unknown. We identified that R. mucilaginosa produced the catechol siderophore enterobactin, the strongest iron-chelating molecule known. We also identified the enterobactin biosynthetic gene cluster (ent-BGC) in global Rothia genomes, which suggests that enterobactin is crucial in Rothia ecology. The purified enterobactin compound impacted growth differentially when amended to cultures of other oral bacterial species. It boosted the growth of commensal Streptococcus salivarius while it reduced the growth of some strains of pathogenic S. mutans. The overarching goal of this study is to determine the role of R. mucilaginosa produced enterobactin in interactions with both the oral microbiota and human oral epithelial cells representing the oral mucosa. We propose an interdisciplinary research approach with two specific aims: Aim 1: Characterization of molecular and ecological responses of oral bacteria to R. mucilaginosa produced enterobactin. The activities of enterobactin will be characterized both in highly diverse oral in vitro grown biofilms, and in bacterial monocultures. The capacity to import enterobactin by different bacterial community members will be revealed by amending growth cultures with fluorescently rhodamine- labeled enterobactin, fluorescence-activated cell sorting, and confocal microscopy. Multi-OMICS sequencing will be conducted to characterize functional changes both in biofilm communities as well as in single and dual- species cultures, specifically targeting genes encoding transport proteins and release mechanisms of enterobactin. Aim 2: Determine the impact of enterobactin on oral mucosal sentinel cells. Co-cultivation systems including both 2D and 3D models of human oral epithelial cells will be applied to study interactions with the oral microbiota and enterobactin. Outcomes of the interactions will be characterized by using a multipronged approach including a comparative gene transcription approach and a gene reporter system that reveals ROS activation in host cells. Production of extracellular pro- and anti-inflammatory cytokines in cell medium relation to enterobactin and oral bacteria will also be addressed to elucidate interactions of importance. The proposed study provides a unique opportunity to expand our knowledge on enterobactin functional role in the oral microbiota, and in interactions with human oral epithelial cells, which is severely lacking. A deeper knowledge of the role of iron scavenging siderophores in the oral cavity will likely bring about a shift in the research field of oral microbial ecology and shine a new light on the importance of iron metabolism in oral health.

项目概要口腔是一个高度多样化的微生物环境，由超过 2000 种细菌、古菌和真菌组成其中大部分物种尚未进行功能表征。大量研究表明，口服 Rothia mucilaginosa 是一种革兰氏阳性口腔共生放线菌，在唾液和牙齿中含量丰富然而，其生态作用尚不清楚。我们发现 R. mucilaginosa 产生了儿茶酚铁载体肠杆菌素，这是已知最强的铁螯合分子。我们还确定了全球 Rothia 基因组中的肠杆菌素生物合成基因簇 (ent-BGC)，这表明肠杆菌素在 Rothia 生态中至关重要。修改后，纯化的肠杆菌素化合物对生长的影响存在差异其他口腔细菌的培养物。它促进了共生唾液链球菌的生长，同时减少了某些致病性变形链球菌菌株的生长。这项研究的总体目标是确定 R. mucilaginosa 产生的肠杆菌素在与口腔微生物群和人类口腔相互作用中的作用代表口腔粘膜的上皮细胞。我们提出了一种跨学科研究方法，其中包括两个具体目标：目标 1：表征口腔细菌对 R 的分子和生态反应。粘液菌产生肠杆菌素。肠杆菌素的活性具有高度多样化的特征口腔体外生长的生物膜和细菌单一培养物。不同来源输入肠杆菌素的能力细菌群落成员将通过用荧光罗丹明修改生长培养物来揭示标记肠杆菌素、荧光激活细胞分选和共聚焦显微镜。多组学测序将进行表征生物膜群落以及单和双膜的功能变化物种培养物，特别针对编码转运蛋白的基因和释放机制肠杆菌素。目标 2：确定肠杆菌素对口腔粘膜前哨细胞的影响。共培养包括人类口腔上皮细胞 2D 和 3D 模型的系统将用于研究与口腔微生物群和肠杆菌素。互动的结果将通过使用多管齐下的方式来表征方法包括比较基因转录方法和揭示 ROS 的基因报告系统宿主细胞中的激活。细胞介质中细胞外促炎和抗炎细胞因子的产生还将讨论肠杆菌素和口腔细菌的相互作用，以阐明重要的相互作用。拟议的研究提供了一个独特的机会来扩展我们对肠杆菌素功能作用的了解。口腔微生物群，以及与人类口腔上皮细胞的相互作用，这是严重缺乏的。更深层次的了解口腔中铁清除铁载体的作用可能会带来改变口腔微生物生态学研究领域，为铁代谢在口腔健康中的重要性提供了新的视角。