Characterizing the basic biology and mechanisms of biofilms in Vibrio cholerae

我的实验室致力于研究霍乱弧菌作为生物膜的基础生物学和作用机制。 模型系统。虽然这种细菌是腹泻疾病霍乱的病原体，但我们并不寻求 研究这种病原体的毒性。相反，我们利用这一成熟的模型系统和基因 我们已经开发了一些工具来描述与生理相关的生物膜的特征。形成了一种 被称为生物膜的复杂的多细胞细菌群落对于毒力、环境 持久性，或不同微生物病原体之间的基因交换。因此，理解这些机制 潜在的生物被膜形成可能揭示抗击各种临床相关感染的新方法。 霍乱弧菌在其水生水库中的甲壳类浮游动物几丁质壳上形成生物膜。 甲壳素生物膜在该生物的生态中扮演着三个重要的角色。首先，霍乱弧菌将甲壳素降解为 可溶性低聚糖，是水环境中重要的碳源和氮源。 其次，甲壳素生物膜的生长诱导自然转化，这是水平基因的一种保守机制。 可以促进获得抗生素耐药基因和新的毒力因子的转移。第三，甲壳素 生物膜对于霍乱的水媒传播很重要。在摄取甲壳素生物膜后，V. 霍乱弧菌必须迅速改变其新陈代谢，从依靠几丁质生长到与肠道微生物群竞争 受感染宿主体内可利用的碳源。我们的模式系统提供了一个独特的机会 描述细菌群落中的细胞如何利用甲壳素的生物表面形成生物膜，作为 营养源，基因交流和传播的平台。在未来五年，我们的目标是定义 甲壳素的初始黏附机制，自然转化过程中DNA的摄取和整合，以及 甲壳素生物膜生长过程中的代谢和传播后哺乳动物宿主中的代谢。 为此，我们产生了一些新的工具来解决这些问题。也就是说，我们使用 一种荧光标记菌毛的新方法，菌毛是最初附着在甲壳素上所需的表面附件 以及在自然转化过程中的DNA摄取。使用这种方法，我们可以观察到的动态性质 这些肉馅。这是任何其他方法都不可能的，应该可以让我们研究它们在甲壳素生物膜中的作用。 菌毛是广泛保守的，我们的研究将解决关于这些的基本和长期存在的问题。 应适用于多种细菌病原体的结构。我们最近还改进了一种方法 用于通过自然转换进行多重基因组编辑(MuGENT)，可用于解剖复合体 基因冗余带来问题的生物学问题。在初步数据中，我们证明了这一点 Tool能够很好地分析霍乱弧菌在甲壳素生物膜和哺乳动物宿主中的代谢。这个 提出的创新方法将为研究关键和保守的过程(即 附着、生物膜形成、自然转化和代谢)在不同微生物物种中。

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.