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

 描述(申请人提供)：为了成功地生存并传播给宿主，兼性致病微生物必须从它们占据的不同的生态位中获得营养。此外，这些营养物质可以作为信号化合物来改变这些微生物的生理。因此，了解病原微生物在其整个生命周期中使用和感受到的营养物质，可以被用来开发新的益生菌、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甲壳素相互作用的分子机制，以及它们在霍乱弧菌在环境宿主中的生存和进化以及传播到人类宿主中的作用。霍乱弧菌是腹泻疾病霍乱的病原体，每年在全世界造成300-500万人感染和10万人死亡。这种兼性病原体是水环境中的常见居民，如果摄入受污染的食物或水就会引起疾病。然而，关于这种病原体在水环境和人类宿主中的代谢仍不清楚。水环境中弧菌的主要碳和氮源是甲壳素，这是甲壳类浮游动物贝壳的主要成分。甲壳素是一种由β1，4连接的N-乙酰氨基葡萄糖(GlcNAc)残基组成的聚合物，是自然界中含量第二丰富的多糖，在水环境中每年产生1011吨。霍乱感染在流行地区是季节性的，与季节性的浮游动物繁殖密切相关。在实验室的水生微宇宙实验中，已经表明甲壳素促进了霍乱弧菌的生长和生物膜的形成，因此可能增强了这种病原体的水传播。许多研究已经证明了利用在富媒体中生长的细菌形成生物膜对霍乱弧菌毒力的重要性。然而，相对较少的研究评估了生长在甲壳素上的生物膜的毒性，甲壳素是水环境中这种病原体最具生理学相关性的营养物质。除了……之外 甲壳素作为一种营养物质，还可以在霍乱弧菌中诱导一种称为自然能力的生理状态，在这种状态下，细菌可以从细胞外环境中摄取DNA。然后，这种DNA可以通过同源重组被分解或整合到基因组中。后一种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，这些基因和突变增强了它们的适应性。通过这种方式，季节性的浮游动物繁殖可能会促进霍乱弧菌在流行地区的快速演变。本研究项目的目的是描述霍乱弧菌与甲壳素相互作用的机制和结果，以及它们在这种病原体通过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，我们建议使用无偏高通量遗传筛选(TN-SEQ)、整体转录组分析(RNA-SEQ)和尖端遗传方法来揭示形成甲壳素生物膜所需的新因素及其相应的调节。在初步工作中，我们已经确定了摄取甲壳素降解产物(甲壳素低聚糖，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菌株和基因报告融合来评估不同甲壳素降解产物的摄取如何影响甲壳素生物膜的空间结构和甲壳素生长的霍乱弧菌的毒力。在目标3中，我们建议评估几丁质诱导霍乱弧菌自然转化的机制。具体地说，我们先前已经证明，在不同的自然能力微生物物种中，在自然转化过程中，能力细菌之间存在表型异质性。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类(FACS)、RNA-SEQ、荧光基因报告程序和互补分子方法来识别与成功的自然转化相关的基因和途径。这些目标将有助于更深入地了解霍乱弧菌如何在水环境中降解、利用和响应甲壳素，并可能发现新的目标和策略，以防止霍乱流行地区的季节性爆发。

 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.