Vibrio cholerae-chitin interactions and their role in cholera transmission and evolution

 描述（由申请人提供）：为了成功存活并传播给宿主，兼性病原微生物必须在它们占据的不同生态位中获得营养。此外，这些营养素可以作为信号化合物来改变这些微生物的生理学。因此，了解病原微生物在其整个生命周期中使用和感知的营养物质可以用于开发新的益生菌，抗菌剂和预防策略。本文提出的研究将揭示霍乱弧菌与代谢产物几丁质相互作用的分子机制，以及它们在这种病原体在其环境水库中的生存和进化以及向人类宿主的传播中的作用。霍乱弧菌（Vibrio cholesterol）是霍乱弧菌的病原体，每年在全世界造成3-5百万感染和> 100，000人死亡。这种兼性病原体是水生环境中的常见居民，如果在受污染的食物或水中摄入就会引起疾病。然而，关于这种病原体在水生环境和人类宿主中的代谢仍不清楚。在水生环境中，弧菌属物种的主要碳源和氮源是多糖几丁质，其是甲壳类浮游动物外壳的主要成分。甲壳素是由β 1，4-连接的N-乙酰葡糖胺（GlcNAc）残基组成的聚合物，并且是自然界中第二丰富的多糖，在水生环境中每年产生>1011吨。霍乱感染在流行地区是季节性的，与浮游动物的季节性繁殖密切相关。在实验室的水生微宇宙实验中，已经表明甲壳素增强霍乱弧菌的生长和生物膜形成，因此，可能增强这种病原体的水传播。许多研究已经证明了生物膜形成对使用在丰富培养基中生长的细菌的霍乱弧菌的毒力的重要性。然而，相对较少的研究，已经评估了生长在甲壳素上的生物膜的毒力，甲壳素是水生环境中这种病原体最具生理相关性的营养物质。除了 除了作为营养物的作用外，几丁质还诱导霍乱弧菌的生理状态，称为天然感受态，其中细菌可以从细胞外环境中摄取DNA。然后，该DNA可以被分解代谢或通过同源重组整合到基因组中。后一个过程被称为自然转化，由不同的微生物物种共享。在进化上，自然转化为微生物提供了一种获得基因和突变的机制，以增强其适应性。以这种方式，浮游动物的季节性水华可能会促进霍乱弧菌在流行地区的快速进化。该研究项目的目标是表征霍乱弧菌-几丁质相互作用的机制和结果及其在该病原体的水传播和进化中的作用。我们制定了三个目标来实现这些目标。在目标1中，我们建议使用无偏见的高通量遗传筛选（Tn-seq），全转录组分析（RNA-seq）和尖端的遗传方法来揭示几丁质生物膜形成及其相应调控所需的新因素。在初步工作中，我们已经确定了吸收几丁质降解产物（几丁质寡糖，GlcNAc和壳寡糖）所需的基因。因此，在目标2中，我们建议使用定义的突变株和基因报告融合来评估不同几丁质降解产物的摄取如何影响几丁质生物膜的空间结构和几丁质生长的霍乱弧菌的毒力。在目标3中，我们提出评估几丁质诱导的霍乱弧菌自然转化的机制。具体来说，我们以前已经表明，在不同的自然能力的微生物物种，有能力的细菌在自然转化的表型异质性。为了解决这种异质性的机制及其在这一保守和关键的进化过程中的作用，我们建议使用荧光激活细胞分选（FACS），RNA-seq，荧光基因报告，和互补的分子方法，以确定基因和途径，与成功的自然转化。这些目标将提供更深入的了解霍乱弧菌如何降解，利用和应对甲壳素在水环境中，并可能发现新的目标和战略，以防止季节性爆发的霍乱流行地区。

 DESCRIPTION (provided by applicant): To successfully survive and transmit to their host, facultative pathogenic microorganisms must acquire nutrients in the diverse niches they occupy. Additionally, these nutrients can serve as signaling compounds to alter the physiology of these microbes. Thus, understanding the nutrients used and sensed by pathogenic microbes throughout their life cycle can be exploited for developing novel probiotics, antimicrobials, and preventative strategies. The research proposed here will uncover the molecular mechanisms underlying the interaction of Vibrio cholerae with the metabolite chitin and their role in the survival and evolution of this pathogen in its environmental reservoir and transmission to its human host. Vibrio cholerae is the causative agent of the diarrheal disease cholera and is annually responsible for 3-5 million infections and >100,000 deaths worldwide. This facultative pathogen is a common resident of the aquatic environment and causes disease if ingested in contaminated food or water. Much about the metabolism of this pathogen in the aquatic environment and the human host, however, remain unclear. A major carbon and nitrogen source for Vibrio species in the aquatic environment is the polysaccharide chitin, which is the primary constituent of the shells of crustacean zooplankton. Chitin is a polymer composed of β1,4- linked N-acetyl glucosamine (GlcNAc) residues, and is the second-most abundant polysaccharide in nature with >1011 tons being produced annually in the aquatic environment. Cholera infections are seasonal in endemic areas and are closely associated with seasonal blooms of zooplankton. In aquatic microcosm experiments in the laboratory, it has been shown that chitin enhances V. cholerae growth and biofilm formation and, therefore, likely enhances the waterborne transmission of this pathogen. Many studies have demonstrated the importance of biofilm formation on the virulence of V. cholerae using bacteria grown in rich media. Relatively few studies, however, have assessed the virulence of biofilms grown on chitin, the most physiologically relevant nutrient for this pathogen in the aquatic environment. In addition to its role as a nutrient, chitin also induces a physiological state in V. cholerae known as natural competence, where bacteria can take up DNA from the extracellular environment. This DNA can then be catabolized or integrated into the genome by homologous recombination. This latter process is known as natural transformation and is shared by diverse microbial species. Evolutionarily, natural transformation provides microorganisms a mechanism for acquiring genes and mutations that enhance their fitness. In this manner, seasonal blooms of zooplankton may promote rapid evolution of V. cholerae in endemic areas. The goals of this research project are to characterize the mechanisms and outcomes of V. cholerae-chitin interactions and their roles in the waterborne transmission and evolution of this pathogen. We have developed three aims to address these goals. In Aim 1, we propose to use unbiased high-throughput genetic screens (Tn-seq), whole transcriptome analysis (RNA-seq), and cutting-edge genetic approaches to uncover novel factors required for the formation of chitin biofilms and their corresponding regulation. In preliminary work we have characterized the genes required for uptake of chitin degradation products (chitin oligosaccharides, GlcNAc, and chitosan oligosaccharides). Thus, in Aim 2, we propose to use defined mutant strains and gene reporter fusions to assess how the uptake of distinct chitin degradation products affects the spatial architecture of a chitin biofilm and the virulence of chitin-grown V. cholerae. In Aim 3, we propose to assess the mechanisms underlying chitin-induced natural transformation in V. cholerae. Specifically, we have previously shown that in diverse naturally competent microbial species, there is phenotypic heterogeneity among competent bacteria during natural transformation. To address the mechanisms underlying this heterogeneity and their role in this conserved and critical evolutionary process we propose to use fluorescence-activated cell sorting (FACS), RNA-seq, fluorescent gene reporters, and complementary molecular methods to identify genes and pathways that correlate with successful natural transformation. These aims will provide a deeper understanding of how V. cholerae degrades, utilizes, and responds to chitin in the aquatic environment, and may uncover novel targets and strategies to prevent seasonal outbreaks of cholera in endemic areas.