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

霍乱弧菌-几丁质相互作用及其在霍乱传播和进化中的作用

基本信息

批准号：
9272812
负责人：
Ankur Dalia
金额：
$ 10.7万
依托单位：
TRUSTEES OF INDIANA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-13 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9272812
关键词：
Address Affect Architecture Area Bacteria Biological Assay Biology Carbohydrates Carbon Catabolism Cells Cessation of life Chitin Chitinase Chitosan Cholera Clinical Competence Crustacea Cyclic AMP Receptor Protein DNA Data Disease Disease Outbreaks Environment Evolution Exhibits Fluorescence Fluorescence Microscopy Fluorescence-Activated Cell Sorting Food Contamination Gene Expression Profile Gene Mutation Genes Genetic Determinism Genetic Screening Genetic Transcription Genome Glucosamine Goals Growth Heterogeneity Horizontal Gene Transfer Human Infection Intervention Laboratories Life Cycle Stages Link Metabolism Methods Microbe Microbial Biofilms Modeling Molecular Mutagenesis Nature Nitrogen Nutrient Oligosaccharides Outcome Pathogenicity Pathway interactions Pattern Phenotype Physiological Physiology Plasticizers Polymers Polysaccharides Population Population Heterogeneity Prevention strategy Probiotics Process Regulation Reporter Reporter Genes Research Research Project Grants Role Signal Transduction Source Surface System Testing Therapeutic Vibrio Vibrio cholerae Virulence Virulence Factors Work Zooplankton antimicrobial base cell transformation combat contaminated water drinking water experimental study extracellular fitness genetic approach homologous recombination microbial microorganism mutant novel novel strategies pathogen prevent public health relevance response sensor histidine kinase transcriptome transcriptome sequencing transmission process uptake waterborne

项目摘要

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.

描述（由适用提供）：为了成功生存并传播给其宿主，兼职致病微生物必须在其占据的潜水细分市场中获取营养。此外，这些营养素可以用作信号化合物，以改变这些微生物的生理。可以探索为开发新颖的益生菌，抗菌剂和预防策略来探索致病微生物所用的营养和感知的营养。此处提出的研究将发现弧菌与代谢物几丁质相互作用的分子机制及其在这种病原体在其环境保护中的生存和演变中的作用，并传播到其人类宿主中。弧菌霍乱是腹泻病霍乱的病因，每年对全球3-500万感染和> 100,000人死亡负责。辅助病原体是水生环境的常见居民，如果被污染的食物或水摄入，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的许多内容尚不清楚。在水生环境中，弧菌物种的主要碳和氮源是多糖几丁质，这是甲壳类烟囱壳的主要组成部分。几丁质是一种由β1,4连接的N-乙酰葡萄糖胺（GlcNAC）保留的聚合物，并且是自然界中第二多的丰富多糖，在水生环境中每年生产> 1011吨。霍乱感染是内在区域的季节性，与浮游动物的季节性血液密切相关。在实验室的水生缩影实验中，已经表明，几丁质增强了霍乱念珠菌的生长和生物膜的形成，因此很可能增强了该病原体的水传播。许多研究表明，使用丰富培养基中生长的细菌，生物膜形成对霍乱弧菌病毒的重要性。然而，相对较少的研究评估了几丁质上生长的生物膜病毒，这是水生环境中这种病原体最相关的营养素。此外几丁质作为营养素的作用还诱导了霍乱弧菌的物理状态，称为自然能力，细菌可以从细胞外环境中吸收DNA。然后可以通过同源重组将该DNA分解代谢或整合到基因组中。以后的过程称为自然转化，被潜水微生物物种共享。从进化上讲，自然转化为获取基因和突变增强其适应性的基因和突变提供了一种机制。以这种方式，浮游动物的季节性血液可以促进霍乱弧菌在地方性地区的快速发展。该研究项目的目标是表征V.霍乱 - 智蛋白相互作用的机制和结果及其在该病原体的水传播和演变中的作用。我们已经开发了三个目标来解决这些目标。在AIM 1中，我们建议使用公正的高通量遗传筛选（TN-SEQ），整个转录组分析（RNA-SEQ）和尖端的遗传方法，以发现形成几丁质生物膜及其相应调节所需的新因素。在初步工作中，我们表征了摄取几丁质降解产物所需的基因（几丁质寡糖，GlcNAC和壳聚糖寡糖）。在AIM 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摄取不同的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壳蛋白生长的V. cholerae的病毒。在AIM 3中，我们建议评估霍乱弧菌中几丁质诱导的自然转化的机制。具体而言，我们以前已经表明，在自然竞争性细菌自然转化过程中，在自然竞争的微生物物种中，在自然转化过程中存在表型异质性。为了解决这种异质性及其在这种保守和关键的进化过程中的作用的机制，我们建议使用荧光激活的细胞分选（FACS），RNA-SEQ，荧光基因报告基因以及互补的分子方法，以鉴定与成功自然转化相关的基因和途径。这些目标将对霍乱谷（V. cholerae）在水生环境中如何降解，利用和响应几丁质的方式有更深入的了解，并可能揭示了防止特有地区霍乱季节性暴发的新颖目标和策略。