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: 10598410
• 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/10598410
• 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; Study models; 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，生物膜对于许多细菌感染的发病机理至关重要。最近， 生物膜也与肠道疾病如结肠直肠癌的发病机制有关， 炎症性肠病，其中从患病肠道中收集的粘膜相关的多微生物群落 在动物模型中结肠恶化疾病。尽管有这些研究，但肠道中生物膜的存在 道一直存在争议。由于肠道内的粘液不断脱落， 层提供用于生物膜形成的稳定表面。目前没有任何可处理的 模型来了解或探索肠道环境中生物膜的形成。因此，证据 缺乏对生物膜形成在粘膜表面定殖中的作用的支持。根据初步数据，我们 显示E.大肠杆菌基因ntrC（ntrC）编码氮利用的调节因子，导致 增加体外生物膜形成，并赋予相对于野生型（WT）E的2- 3log竞争优势。杆菌 小鼠体内（尽管体外生长速率降低）。这些结果与生物膜形成的作用一致 在肠道内的殖民化。因此，我假设，竞争健身的ntrC E。大肠杆菌是生物膜的反映 在肠道的粘膜表面形成。其目的是进一步描述 E.大肠杆菌定植的肠道，并使用这个模型系统，以了解更多 通常，生物膜在肠粘膜界面的疾病发病机制中的作用。这一假设将 通过三个相互关联的具体目标进行测试，这些目标将确定生物膜形成背后的关系 体外和空间定位的定植在整个小鼠小肠和结肠（目的1），确定 铁在E.大肠杆菌生物膜形成和肠道定植（目的2），并表征生物学特性 使用结肠炎小鼠模型研究细菌生物膜形成在疾病发病机制中的影响（目的3）。的 实验将使用各种创新的方法，包括创造和接种遗传- 修饰的大肠大肠杆菌菌株，通过ICP-MS定量肠粘液和管腔中的铁，以及16 S rRNA 基因微生物群测序来解决这些生理相关机制。拟议的研究是 重要的是，它将提供有关生物膜形成的细菌决定因素的新信息， 肠道，与使用它们在结肠炎模型中的竞争性生态位排除的理由。大学 宾夕法尼亚州提供了完美的研究环境，进行这项调查，因为当地的专业知识， 肠道微生物组和基础微生物学的相互关联领域。候选人将获得以下方面的经验： 微生物组的组成和功能分析，获得微生物基因的基本技能 工程技术，并扩大她的微生物学知识基础。这些技能将大大 加强候选人的职业发展成为一个独立的科学调查员，与长期 目标是使我们能够更好地了解和调节肠道微生物组在疾病状态下的作用。