Small RNAs and Vibrio cholerae adaptation to different carbon sources

描述（由申请人提供）：霍乱是一种由霍乱弧菌引起的毁灭性腹泻疾病，霍乱弧菌是一种水生病原体。霍乱弧菌在其水生宿主和人类宿主之间转换时，必须适应不断变化的营养物质供应，如碳源。转录分析和诱变筛选先前表明，能量代谢途径，特别是磷酸烯醇丙酮酸(PEP)-碳水化合物磷酸转移酶系统（PTS），在霍乱弧菌在不同生态位之间的转变中起主要作用。然而，霍乱弧菌PTS的成分在很大程度上仍未确定。本研究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。许多小rna （sRNAs）参与霍乱弧菌和相关细菌的应激适应的多种调控途径。本研究的目的是确定srna介导的机制是否参与了霍乱弧菌对不同碳源的适应。先前的研究已经确定了霍乱弧菌转录组中的sRNAs可能参与调节这种兼性病原体的PTS。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体mtlA的表达。核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，从而有助于其环境持久性。中心假设将通过追求三个具体目标来检验。首先，确定srna介导的甘露醇代谢调控机制。利用生物化学方法和诱变技术，阐明MtlS对mtlA表达的影响。本提案的第二个目标将使用双重遗传选择来确定mtl靶向的最小mtlA调控基序。本提案的第三个目的是通过生化分析和诱变来确定mtlS表达的调节因子。本文提出的实验将促进对sRNAs在碳代谢中的作用的理解，并阐明可能为霍乱弧菌疫苗和治疗提供靶点的中心代谢途径的组成部分。因此，拟议的研究与NIH开发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良好模型；因此，这一建议也将提供对其他已知和新出现的病原体的见解。

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.