Small RNAs and Vibrio cholerae adaptation to different carbon sources

小RNA和霍乱弧菌对不同碳源的适应

• 批准号: 8758751
• 负责人: Jane May Liu
• 金额: $ 39.53万
• 依托单位: POMONA COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-15 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8758751
• 关键词: 5' Untranslated Regions; Address; Algae; Back; Bacteria; Behavioral; Binding; Biochemical; Bioinformatics; Biological Assay; Biology; Carbohydrates; Carbon; Catabolism; Cessation of life; Cholera; Cholera Toxin; Complex; Data; Development; Disease; Elements; Environment; Eukaryota; Fructose; Gastrointestinal tract structure; Genes; Genetic; Genetic Screening; Genome; Goals; Hand; Human; Ingestion; Intestines; Knowledge; LacZ Genes; Lead; Life Cycle Stages; Liquid substance; Mannitol; Marines; Mass Spectrum Analysis; Messenger RNA; Metabolic; Metabolism; Mission; Operon; Outcome; Pathway interactions; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphotransferases; Physiology; Process; Production; Promoter Regions; Protein Biosynthesis; Proteins; Proteomics; Public Health; Regulation; Regulatory Pathway; Research; Sanitation; Signal Transduction; Small Intestines; Small RNA; Source; Study models; Sugar Alcohols; System; Testing; Toxin; Vaccines; Vibrio cholerae; Water; Work; antimicrobial drug; base; combat; environmental change; in vivo; innovation; insight; novel therapeutics; novel vaccines; pathogen; permease; public health relevance; research study; response; therapeutic target; transcription factor; waterborne

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how Vibrio cholerae is able to rapidly adapt to and multiply within both aquatic environments and the host gastrointestinal tract. Understanding the entire life cycle of V. cholerae, specifically how it modulates its physiology to adjust to differet environments, is critical to enhancing combat against this pathogen. The long-term goal of this research is to understand how V. cholerae adapts to different carbon sources and thereby persists in varying niches. The objective of this application is to identify and characterize genetc mechanisms by which V. cholerae synchronizes its physiology with available carbon sources. The central hypothesis of this proposal is that V. cholerae relies on a suite of small regulatory RNAs (sRNAs) to adjust to changing environmental conditions. There is growing evidence that in many bacteria, including V. cholerae, sRNA regulatory circuits modulate metabolic and behavioral processes. Thus, to truly understand how V. cholerae is able to adjust its physiology for long-term persistence, the multiple sRNAs involved must be identified and characterized. This project will address this need through three specific aims: 1) Determine how expression of the sRNA MtlS is regulated; 2) Characterize the mechanism through which the mannitol permease, MtlA, is degraded when mannitol is no longer present; and 3) Determine the in vivo function of the sRNA IGR4. Under the first aim, the regulation of an sRNA involved in mannitol metabolism, MtlS, will be studied using a transcriptional fusion between the mtlS promoter region and lacZ; genetic screens will lead to the identification and characterization of regulators of mtlS expression. Under the second aim, mass spectrometry-based quantitative proteomics will be used to identify proteins that are up-regulated upon expression of MtlS. These proteins will then be characterized with regard to their effect on the stability of the mannitol transporter MtlA. Under the third aim, biochemical assays will be used to test the working hypothesis that the sRNA IGR4 down-regulates synthesis of the Cra protein, a repressor of many glycolytic genes, including mtlA. Preliminary data support the working hypotheses in this application and establish that the proposed experiments are feasible in the applicant's hands. The proposed research is significant because it will identify components of carbon acquisition and metabolism that may inform the development of new therapeutics or vaccines that are more effective than those currently available. Indeed, the phosphoenolpyruvate:sugar phosphotransferase system (PTS) - the primary transporter of many carbohydrates in bacteria and the major system that will be studied in this proposal - is absent from eukaryotes and has been described as an ideal target for antimicrobial agents. The approach is innovative because it seeks to investigate the persistence of V. cholerae by defining new regulatory circuits that control the PTS in this pathogen.

描述（由申请人提供）： 在了解霍乱弧菌如何能够在水生环境和宿主胃肠道中快速适应和繁殖方面存在根本差距。了解霍乱弧菌的整个生命周期，特别是它如何调节其生理机能以适应不同的环境，对于加强对抗这种病原体至关重要。这项研究的长期目标是了解霍乱弧菌如何适应不同的碳源，从而在不同的生态位中持续存在。本应用的目的是确定和表征霍乱弧菌将其生理学与可用碳源同步的基因机制。该提案的核心假设是霍乱弧菌依赖一套小调节 RNA (sRNA) 来适应不断变化的环境条件。越来越多的证据表明，在包括霍乱弧菌在内的许多细菌中，sRNA 调节回路可调节代谢和行为过程。因此，为了真正了解霍乱弧菌如何调整其生理机能以实现长期持续存在，必须对所涉及的多种 sRNA 进行鉴定和表征。该项目将通过三个具体目标来满足这一需求： 1) 确定 sRNA MtlS 的表达是如何调节的； 2) 描述当甘露醇不再存在时甘露醇渗透酶 MtlA 被降解的机制； 3)确定sRNA IGR4的体内功能。第一个目标是利用 mtlS 启动子区域和 lacZ 之间的转录融合来研究参与甘露醇代谢的 sRNA MtlS 的调节；基因筛选将导致监管者的识别和表征 mtlS 表达。第二个目标是，基于质谱的定量蛋白质组学将用于鉴定 MtlS 表达上调的蛋白质。然后将表征这些蛋白质对甘露醇转运蛋白 MtlA 稳定性的影响。在第三个目标下，生化检测将用于测试 sRNA IGR4 下调 Cra 蛋白合成的工作假设，Cra 蛋白是许多糖酵解基因（包括 mtlA）的抑制因子。初步数据支持本申请中的工作假设，并证明所提出的实验在申请人手中是可行的。拟议的研究意义重大，因为它将确定碳获取和代谢的组成部分，这可能有助于开发比目前可用的更有效的新疗法或疫苗。事实上，磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）——细菌中许多碳水化合物的主要转运蛋白，也是本提案中将研究的主要系统——在真核生物中不存在，并且被描述为抗菌剂的理想靶点。该方法具有创新性，因为它试图通过定义控制霍乱弧菌病原体中 PTS 的新调控回路来研究霍乱弧菌的持久性。