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.

项目摘要口腔是一个高度多样的微生物环境，由> 2000个细菌，古细菌和真菌组成物种大多数尚未在功能上表征。许多研究已经确定了口头 Rothia Mucilaginosa是一种革兰氏阳性的口服分子性静脉细菌，在唾液和牙齿中高度丰富然而，在全球人口中，其生态作用尚不清楚。我们确定了粘麦那菌产生了Catechol铁载体肠乳素，这是已知的最强铁授予分子。我们也确定了全球Rothia基因组中的肠乳素生物合成基因簇（ENT-BGC），这表明肠actin 在Rothia生态学中至关重要。修订后，纯化的肠霉素化合物会差异地影响生长到其他口腔细菌的培养物。它提高了共生链球菌唾液的生长减少了一些致病性链球菌的生长。这项研究的总体目标是确定粘霉菌菌的作用在与口服微生物群和人口腔的相互作用中产生肠骨动物代表口服粘膜的上皮细胞。我们提出了一种跨学科研究方法具体目的：目标1：口服细菌对R的分子和生态反应的表征。粘膜胶质可产生肠乳蛋白。肠霉素的活动将在高度多样化中表征口服体外生长的生物膜和细菌单栽培。通过不同的能力导入肠药细菌社区成员将通过修改荧光淡褐色的生长培养物来揭示标记的肠霉素，荧光激活的细胞分选和共聚焦显微镜。多词测序将进行以表征生物膜群落以及单一和双重的功能变化物种培养物，专门针对编码转运蛋白的基因和释放机制肠毒素。 AIM 2：确定肠霉素对口服粘膜前哨细胞的影响。共培养包括人口腔上皮细胞的2D和3D模型在内的系统将应用于研究与口服微生物群和肠霉素。相互作用的结果将通过使用多收益来表征包括比较基因转录方法和揭示ROS的基因报告基因系统的方法宿主细胞中的激活。细胞培养基关系中细胞外促和抗炎细胞因子的产生肠球菌和口服细菌也将被解决以阐明重要性的相互作用。拟议的研究提供了一个独特的机会，可以扩大我们对肠乳蛋白功能作用的知识口服微生物群，以及与人口腔上皮细胞的相互作用，严重缺乏。更深了解铁清除铁载体在口腔中的作用可能会带来变化口腔微生物生态学的研究领域，并阐明了铁代谢在口腔健康中的重要性。