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（由申请人提供）：为了成功生存并传播给宿主，兼性病原微生物必须在其占据的不同生态位中获取营养物。此外，这些营养物可以作为信号化合物来改变这些微生物的生理学。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用于开发新型益生菌、抗菌剂和预防策略，本文提出的研究将揭示它们之间相互作用的分子机制。霍乱弧菌及其代谢物几丁质及其在该病原体在其环境储存库中的生存和进化以及向人类宿主传播中的作用霍乱弧菌是腹泻病霍乱的病原体，每年导致 3-500 万人感染和传播。全球超过 100,000 人死亡。这种兼性病原体是水生环境中的常见居民，如果摄入受污染的食物或水中，就会导致疾病。然而，水生环境中弧菌物种的主要碳源和氮源是多糖甲壳素，它是甲壳类浮游动物壳的主要成分，甲壳素是由 β1 组成的聚合物。 4-连接的 N-乙酰氨基葡萄糖 (GlcNAc) 残基，是自然界中第二丰富的多糖，每年生产超过 1011 吨水生环境中的霍乱感染是季节性的，并且与浮游动物的季节性繁殖密切相关。实验室的水生微观实验表明，甲壳素可促进霍乱弧菌的生长和生物膜的形成，因此可能会增强水传播。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毒力的重要性，然而，评估了这种病原体的传播。甲壳素上生长的生物膜的毒力，甲壳质是水生环境中与该病原体最生理相关的营养物质。作为营养物质，几丁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吸收 DNA，然后通过同源重组将这种 DNA 分解代谢或整合到基因组中。被称为自然转化，是多种微生物物种共有的。在进化上，自然转化为微生物提供了一种获取基因和突变的机制，从而增强了它们的适应性。通过这种方式，浮游动物的季节性繁殖可以促进快速进化。该研究项目的目标是确定霍乱弧菌与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，我们建议使用无偏倚的高通量遗传筛选 (Tn-seq)、全转录组分析 (RNA-seq) 和尖端遗传方法来发现几丁质形成所需的新因子在初步工作中，我们已经表征了摄取几丁质降解产物（几丁质寡糖、GlcNAc 和壳聚糖寡糖）所需的基因，因此，在目标 2 中，我们建议使用确定的突变菌株和基因报告融合体。在目标 3 中，我们评估不同甲壳素降解产物的吸收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。提出评估霍乱弧菌中几丁质诱导的自然转化的机制。具体来说，我们之前已经证明，在自然多样化的感受态微生物物种中，感受态细菌在自然转化过程中存在表型异质性。为了在这一保守且关键的进化过程中发挥作用，我们建议使用荧光激活细胞分选（FACS）、RNA-seq、荧光基因产生器和补充分子方法来识别与成功自然转化相关的基因和途径。更深入地了解霍乱弧菌如何降解、利用和响应水生环境中的几丁质，并可能发现预防霍乱流行地区季节性爆发的新目标和策略。