Characterizing the basic biology and mechanisms of biofilms in Vibrio cholerae

霍乱弧菌生物膜的基本生物学和机制特征

• 批准号: 10468749
• 负责人: Ankur Dalia
• 金额: $ 38.7万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10468749
• 关键词: Address; Adherence; Antibiotic Resistance; Bacteria; Biological; Biological Models; Biology; Carbon; Cells; Chitin; Cholera; Complex; Crustacea; DNA; Data; Ecology; Environment; Genetic; Growth; Horizontal Gene Transfer; Infection; Ingestion; Label; Metabolism; Methods; Microbial Biofilms; Nature; Nitrogen; Nutrient; Oligosaccharides; Organism; Physiological; Pilum; Play; Process; Role; Source; Structure; Surface; Vibrio cholerae; Virulence; Virulence Factors; Zooplankton; appendage; bacterial community; clinically relevant; combat; diarrheal disease; genome editing; gut microbiota; improved; innovation; microbial; novel; novel strategies; pathogen; pathogenic bacteria; pathogenic microbe; resistance gene; stem; tool; transmission process; uptake; waterborne

My lab is focused on studying the basic biology and mechanisms of biofilms using Vibrio cholerae as a model system. While this bacterium is the causative agent of the diarrheal disease cholera, we do not seek to study the virulence of this pathogen. Instead, we leverage this well-established model system and the genetic tools we have developed to characterize biofilms in a physiologically relevant context. The formation of complex multicellular bacterial communities known as biofilms is critical for virulence, environmental persistence, or genetic exchange in diverse microbial pathogens. Thus, understanding the mechanisms underlying biofilm formation may uncover novel approaches to combat diverse clinically relevant infections. V. cholerae forms biofilms in its aquatic reservoir on the chitinous shells of crustacean zooplankton. Chitin biofilms play three important roles in the ecology of this organism. First, V. cholerae degrades chitin into soluble oligosaccharides, which serve as an important carbon and nitrogen source in the aquatic environment. Second, growth in chitin biofilms induces natural transformation, a conserved mechanism of horizontal gene transfer that can promote the acquisition of antibiotic resistance genes and novel virulence factors. Third, chitin biofilms are important for the waterborne transmission of cholera. Following ingestion of a chitin biofilm, V. cholerae must rapidly alter its metabolism from growth on chitin to competing with the intestinal microbiota for the carbon sources available within its infected host. Our model system provides a unique opportunity to characterize how cells within a bacterial community utilize the biotic surface of chitin for biofilm formation, as a nutrient source, a platform for genetic exchange, and transmission. In the next five years, we aim to define the mechanisms of initial adherence to chitin, DNA uptake and integration during natural transformation, and metabolism during growth in chitin biofilms and in the mammalian host following transmission. To that end, we have generated a number of novel tools to address these questions. Namely, we use a novel method to fluorescently label pili, which are surface appendages required for initial attachment to chitin and for DNA uptake during natural transformation. Using this method, we can observe the dynamic nature of these pili. This is not possible by any other approach and should allow us to address their role in chitin biofilms. Pili are broadly conserved, and our studies will address fundamental and long-standing questions about these structures that should be applicable to many bacterial pathogens. We have also recently improved a method for multiplex genome editing by natural transformation (MuGENT), which can be used to dissect complex biological questions where genetic redundancy poses an issue. In preliminary data we demonstrate that this tool is well poised to dissect the metabolism of V. cholerae in chitin biofilms and in the mammalian host. The innovative approaches proposed will provide a paradigm for the study of critical and conserved processes (i.e. adherence, biofilm formation, natural transformation, and metabolism) in diverse microbial species.

我的实验室专注于使用霍乱弧菌作为生物膜的基础生物学和机制研究 模型系统。虽然这种细菌是腹泻病霍乱的病原体，但我们并不寻求 研究这种病原体的毒力。相反，我们利用这个完善的模型系统和遗传 我们开发的工具用于在生理相关背景下表征生物膜。的形成 复杂的多细胞细菌群落（称为生物膜）对于毒力、环境和环境至关重要 不同微生物病原体的持久性或基因交换。因此，了解机制 潜在的生物膜形成可能会揭示对抗各种临床相关感染的新方法。 霍乱弧菌在甲壳类浮游动物的几丁质壳上的水生水库中形成生物膜。 几丁质生物膜在该生物体的生态学中发挥三个重要作用。首先，霍乱弧菌将几丁质降解为 可溶性低聚糖，是水生环境中重要的碳源和氮源。 其次，几丁质生物膜的生长诱导自然转化，这是水平基因的保守机制 可以促进抗生素抗性基因和新毒力因子获得的转移。三、甲壳素 生物膜对于霍乱的水传播非常重要。摄入几丁质生物膜后，V. 霍乱弧菌必须迅速改变其代谢，从在几丁质上生长转变为与肠道微生物群竞争 受感染宿主体内可用的碳源。我们的模型系统提供了一个独特的机会 描述细菌群落内的细胞如何利用几丁质的生物表面形成生物膜，作为 营养源，基因交换和传播的平台。未来五年，我们的目标是定义 自然转化过程中初始粘附几丁质、DNA 摄取和整合的机制，以及 几丁质生物膜生长期间和传播后哺乳动物宿主中的代谢。 为此，我们开发了许多新颖的工具来解决这些问题。也就是说，我们使用一个 荧光标记菌毛的新方法，菌毛是初始附着到甲壳素所需的表面附属物 以及自然转化过程中 DNA 的摄取。使用这种方法，我们可以观察到动态性质 这些霹雳。这是任何其他方法都不可能实现的，并且应该使我们能够解决它们在几丁质生物膜中的作用。 Pili 广泛保守，我们的研究将解决有关这些的基本和长期存在的问题 应该适用于许多细菌病原体的结构。我们最近还改进了一个方法 通过自然转化进行多重基因组编辑（MuGENT），可用于剖析复杂的基因组 遗传冗余引起问题的生物学问题。在初步数据中，我们证明了这一点 该工具已准备好剖析霍乱弧菌在几丁质生物膜和哺乳动物宿主中的代谢。这 提出的创新方法将为研究关键和保守过程（即 不同微生物物种的粘附、生物膜形成、自然转化和代谢）。