Small RNAs and Vibrio cholerae adaptation to different carbon sources

描述(由申请人提供)： 在理解霍乱弧菌如何能够在水生环境和宿主胃肠道内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，特别是它如何调节自己的生理以适应不同的环境，对于加强对这种病原体的抗击至关重要。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。本申请的目的是确定和表征霍乱弧菌与可用碳源同步其生理的基因ETC机制。这一提议的中心假设是霍乱弧菌依赖一套小的调节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调节回路调节新陈代谢和行为过程。因此，为了真正了解霍乱弧菌如何能够调整其生理学以适应长期的持久性，必须识别和表征涉及的多个sRNA。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)表征当甘露醇不再存在时甘露醇渗透酶mtlA被降解的机制；以及3)确定sRNA IGR4的体内功能。在第一个目标下，将通过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sna的调节；遗传筛选将导致对调节因子的鉴定和表征。 MtlS的表达。在第二个目标下，基于质谱学的定量蛋白质组学将被用来识别MtlS表达上调的蛋白质。然后，将根据它们对甘露醇转运体MtlA稳定性的影响来表征这些蛋白质。在第三个目标下，将使用生化分析来检验工作假设，即sRNA IGR4下调CRA蛋白的合成，CRA蛋白是包括mtlA在内的许多糖酵解基因的阻遏物。初步数据支持本申请中的工作假设，并证明拟议的实验在申请人手中是可行的。这项拟议的研究意义重大，因为它将确定碳获取和新陈代谢的成分，这些成分可能会为开发比目前可用的更有效的新疗法或疫苗提供信息。事实上，磷酸烯醇式丙酮酸：糖磷酸转移酶系统(PTS)-细菌中许多碳水化合物的主要转运体，也是本提案中将研究的主要系统-在真核生物中不存在，已被描述为抗菌剂的理想靶标。这种方法是创新的，因为它试图通过定义控制霍乱弧菌中PTS的新调节电路来研究霍乱弧菌的持久性。

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.