Small RNAs and Vibrio cholerae adaptation to different carbon sources

描述(申请人提供)：霍乱是一种毁灭性的腹泻疾病，由霍乱弧菌引起，霍乱弧菌是一种由水传播的病原体。霍乱弧菌在其水生宿主和人类宿主之间过渡时，必须适应不断变化的营养可获得性，如碳源。转录分析和诱变筛选表明，能量代谢途径，特别是磷酸烯醇式丙酮酸(PEP)-碳水化合物磷酸转移酶系统(PTS)，在霍乱弧菌不同生态位之间的转换中起着重要作用。然而，霍乱弧菌PTS的成分在很大程度上仍不清楚。这项建议的长期目标是阐明霍乱弧菌如何调节PTS以适应不断变化的环境。在霍乱弧菌和相关细菌中，许多小RNA(SRNAs)参与了不同的调节途径，涉及应激适应。这项建议的目的是确定是否有sRNA介导的机制参与霍乱弧菌对不同碳源的适应。以前的研究已经在霍乱弧菌转录组中发现了可能参与调节这种兼性病原体PTS的sRNAs。强有力的初步数据表明，SRNA mtlS以一种受调控的方式表达，并抑制霍乱弧菌甘露醇特异性转运蛋白mtlA的表达。中心假设是霍乱弧菌利用MtlS介导的基因表达控制来快速适应碳源的变化，从而有助于其环境的持久性。核心假设将通过追求三个具体目标来检验。首先，将确定sRNA介导的甘露醇代谢调节的机制。利用生化方法和诱变方法，将阐明mtlS对mtlA表达的影响。该提案的第二个目标将使用双重遗传选择来确定MTLS靶向的最小mtlA调控基序。这项建议的第三个目标是利用生化分析和诱变来确定mtlS表达的调节因子。这里提出的实验将促进对sRNA在碳代谢中作用的理解，并阐明可能为霍乱弧菌疫苗和治疗提供靶点的中央代谢途径的组成部分。因此，这项拟议的研究与美国国立卫生研究院发展基础知识以减轻人类疾病负担的使命相关。此外，霍乱弧菌是其他兼性细菌的极好模型；因此，这一建议也将为其他已知和新出现的病原体提供洞察力。 公共卫生相关性：该项目涉及确定霍乱的病原体霍乱弧菌如何适应不同的环境。据估计，每年有100多万病例，霍乱是一个主要的全球公共卫生问题。了解霍乱弧菌在水生环境和宿主之间过渡时所经历的生化和生理变化，将为我们如何使用下一代疗法和疫苗对抗这些病原体提供见解。

DESCRIPTION (provided by applicant): Cholera is a devastating diarrheal disease caused by Vibrio cholerae, a water-born pathogen. V. cholerae must adapt to changing nutrient availability, such as carbon sources, when transitioning between its aquatic reservoirs and the human host. Transcriptional analysis and mutagenesis screens have previously indicated that energy metabolism pathways, particularly the phosphoenolpyruvate (PEP)-carbohydrate phosphotransferase system (PTS), play a major role in the transition of V. cholerae between different niches. However, the components of the V. cholerae PTS remain largely uncharacterized. The long-term goal of this proposal is to elucidate how V. cholerae regulates the PTS to adapt to changing environments. Many small RNAs (sRNAs) are involved in diverse regulatory pathways involving stress-adaptation in V. cholerae and related bacteria. The objective of this proposal is to determine if an sRNA-mediated mechanism is involved in V. cholerae adaptation to different carbon sources. Previous studies have identified sRNAs in the V. cholerae transcriptome that may be involved in regulating the PTS of this facultative pathogen. Strong preliminary data suggests that an sRNA, MtlS, is expressed in a regulated manner and inhibits the expression of mtlA, the mannitol- specific transporter of V. cholerae. The central hypothesis is that V. cholerae uses MtlS-mediated control of gene expression to rapidly adapt to changes in carbon source, contributing to its environmental persistence. The central hypothesis will be tested by pursuing three specific aims. First, the mechanism of sRNA-mediated regulation of mannitol metabolism will be determined. Using biochemical approaches and mutagenesis, effects of MtlS on mtlA expression will be elucidated. The second aim of this proposal will use dual genetic selections to identify minimal mtlA regulatory motifs targeted by MtlS. The third aim of this proposal is to identify the regulator of mtlS expression using biochemical assays and mutagenesis. The experiments proposed here will advance the understanding of the role of sRNAs in carbon metabolism and elucidate components of a central metabolic pathway that may provide targets for vaccines and therapeutics against V. cholerae. Thus, the proposed research is relevant to the NIH's mission of developing fundamental knowledge that will reduce the burdens of human diseases. Furthermore, V. cholerae is an excellent model for other facultative bacteria; thus, this proposal will also provide insight into other known and emerging pathogens. PUBLIC HEALTH RELEVANCE: This project involves determining how the bacterial pathogen Vibrio cholerae, the causative agent of cholera, adapts to different environments. With an estimated greater than one million cases each year, cholera represents a major global public health concern. Understanding the biochemical and physiological changes V. cholerae undergoes while transitioning between aquatic environment and host will provide insights into how we may combat these pathogens with next-generation therapeutics and vaccines.