Determinants of bacterial biofilm formation at the intestinal mucosal interface and their roles in pathogen exclusion

肠粘膜界面细菌生物膜形成的决定因素及其在病原体排除中的作用

• 批准号: 10612990
• 负责人: Josephine Ni
• 金额: $ 16.04万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612990
• 关键词: Address; Anabolism; Animal Model; Bacteria; Bacterial Infections; Biologic Characteristic; Biological; Biological Assay; Biological Models; Body Weight decreased; Cell Adhesion; Characteristics; Chelating Agents; Chemicals; Citrobacter rodentium; Colitis; Colon; Colonic Diseases; Colorectal Cancer; Communities; Complement; Complex; Confocal Microscopy; Data; Dental caries; Dietary Iron; Disease; Engineered Gene; Engineering; Enterobactin; Environment; Escherichia coli; Exclusion; Genes; Goals; Growth; Harvest; Image; Immunofluorescence Immunologic; In Vitro; Inductively Coupled Plasma Mass Spectrometry; Infection; Infectious colitis; Inflammatory Bowel Diseases; Intestinal Diseases; Intestinal Mucosa; Intestines; Investigation; Iron; Knowledge; Length; Link; Microbial Biofilms; Microbiology; Modeling; Mucins; Mucous Membrane; Mucous body substance; Mus; Mutation; Nitrogen Fixation Genes; Nutrient; Pathogenesis; Pathway interactions; Pattern; Pennsylvania; Pentetic Acid; Physiological; Pilum; Play; Proteins; Proteomics; Radial; Research; Research Personnel; Role; Small Intestines; Specificity; Surface; Techniques; Testing; Universities; Urinary tract; career development; experience; experimental study; fitness; gain of function; gut colonization; gut microbiome; in vivo; inhibitor; innovation; iron supplementation; knowledge base; loss of function; microbial; microbiome analysis; microbiota; mucosal microbiota; murine colitis; mutant; pathogen; rRNA Genes; skills

Project Summary It is well established that biofilms are critical for the pathogenesis of many bacterial infections. More recently, biofilms have also been implicated in the pathogenesis of intestinal diseases such as colorectal cancer and inflammatory bowel disease, where mucosal-associated polymicrobial communities harvested from diseased colon exacerbates disease in animal models. Despite these studies, the presence of biofilms in the intestinal tract has been controversial. Since the mucus that lines the intestinal tract is constantly shed, the ability of this layer to provide a stable surface for biofilm formation has been questioned. There are currently no tractable models to understand or explore biofilm formation in the intestinal environment. As a result, evidence supporting a role for biofilm formation in colonization of the mucosal surface is lacking. In preliminary data, we show that deletion of the E. coli gene ntrC (∆ntrC), which encodes a regulator for nitrogen utilization, results in increased biofilm formation in vitro and confers a 2-3 log competitive advantage over wild-type (WT) E. coli in vivo in mice (despite reduced growth rate in vitro). These results are consistent with a role for biofilm formation in intestinal colonization. I therefore hypothesize that competitive fitness of ∆ntrC E. coli is a reflection of biofilm formation at the mucosal surface of the intestinal tract. The objective is to further characterize the role of biofilm formation in E. coli colonization of the intestinal tract and to use this model system to understand more generally the role of biofilms in disease pathogenesis at the intestinal mucosal interface. This hypothesis will be tested through three inter-related specific aims that will determine the relationship behind biofilm formation in vitro and spatially-localized colonization throughout the murine small intestine and colon (Aim 1), determine the role of iron in E. coli biofilm formation and intestinal colonization (Aim 2), and characterize the biological impact of bacterial biofilm formation in disease pathogenesis using a murine model of colitis (Aim 3). The experiments will use various innovative approaches including the creation and inoculation of genetically- modified E. coli strains, quantification of iron via ICP-MS in the intestinal mucus and lumen, and 16S rRNA gene microbiota sequencing to address these physiologically-relevant mechanisms. The proposed research is significant because it will provide new information about the bacterial determinants of biofilm formation in the intestinal tract, with the rationale of using them for competitive niche exclusion in models of colitis. University of Pennsylvania provides the perfect research environment to conduct this investigation given local expertise in the inter-related fields of gut microbiome and basic microbiology. The candidate will gain experience in compositional and functional analysis of the microbiome, acquire fundamental skills in microbial gene engineering techniques, and broaden her knowledge base of microbiology. These skill sets will greatly enhance the candidate's career development into an independent scientific investigator, with the long-term goal of enabling us to better understand and modulate the effects of the gut microbiome in disease states.

项目摘要 众所周知，生物膜在许多细菌感染的发病机制中起着关键作用。最近， 生物膜也与肠道疾病的发病机制有关，如结直肠癌和 炎症性肠病，其中黏膜相关的多菌群落从疾病中收获 在动物模型中，结肠会加重疾病。尽管有这些研究，但肠道中生物膜的存在 TRACT一直备受争议。由于肠道中的粘液不断地排出，这一能力 为生物膜的形成提供稳定表面的层一直受到质疑。目前还没有可驯服的 了解或探索肠道环境中生物膜形成的模型。因此，证据 支持生物膜形成在粘膜表面定植中的作用是缺乏的。在初步数据中，我们 研究表明，编码氮素利用调节器大肠杆菌基因ntrc(∆ntrc)的缺失导致 增加了体外生物被膜的形成，使其比野生型(WT)大肠杆菌具有2-3对数竞争优势 在小鼠体内(尽管体外生长速度降低)。这些结果与生物膜形成的作用是一致的。 在肠道定植中。因此，我假设∆ntrc大肠杆菌的竞争适合性是生物被膜的反映。 在肠道的粘膜表面形成。我们的目标是进一步描述 大肠埃希氏菌在肠道定植中生物膜的形成及利用该模型系统了解更多 一般来说，生物膜在肠道粘膜界面的疾病发病机制中的作用。这一假说将 通过三个相互关联的具体目标进行测试，这三个目标将决定生物膜形成背后的关系 在小鼠小肠和结肠的体外和空间定位定植(目标1)，确定 铁在大肠杆菌生物膜形成和肠道定植中的作用(目标2)，并表征了生物 利用小鼠结肠炎模型研究细菌生物膜形成在疾病发病机制中的作用(目标3)。这个 实验将使用各种创新方法，包括创造和接种基因- 改良的大肠杆菌菌株，通过电感耦合等离子体质谱定量测定肠道粘液和肠腔中的铁，以及16S rRNA 基因微生物群测序，以解决这些生理相关的机制。拟议的研究是 意义重大，因为它将提供关于生物膜形成的细菌决定因素的新信息 肠道，其理论基础是在结肠炎模型中使用它们进行竞争性生态位排斥。中华人民共和国大学 考虑到当地的专业知识，宾夕法尼亚州提供了进行这项调查的完美研究环境 肠道微生物组和基础微生物学相互关联的领域。候选人将在以下方面获得经验 微生物组的组成和功能分析，掌握微生物基因的基本技能 工程技术，并拓宽了她的微生物学知识基础。这些技能组合将极大地 增强应聘者的职业发展，成为一名独立的科学调查员，具有长期的 目标是使我们能够更好地了解和调节肠道微生物群在疾病状态下的影响。