Metabolic control of the second messenger cyclic diguanylate: implications to intracellular bacterial pathogens

第二信使环二鸟苷酸的代谢控制：对细胞内细菌病原体的影响

• 批准号: 10055808
• 负责人: Mauricio Henriques Pontes
• 金额: $ 23.75万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-21 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10055808
• 关键词: Acute; Affect; Anabolism; Bacteria; Behavior; Biological Assay; Cell physiology; Cells; Cellulose; Communities; Cues; Defect; Development; Enzymes; Extracellular Matrix; Fluorescence; Fostering; GMP synthase; Genes; Genetic Epistasis; Goals; Growth; High-Throughput Nucleotide Sequencing; Infection; Investigation; Life Style; Maps; Mediating; Metabolic; Metabolic Control; Metabolism; Microbial Biofilms; Mus; Mutation; Organism; Pathway interactions; Periodicity; Phagocytes; Physiological; Physiological Adaptation; Play; Production; Protein Biosynthesis; Proteins; Proxy; Pyruvate Kinase; Reporter; Reporting; Ribosomes; Role; Salmonella; Salmonella typhimurium; Second Messenger Systems; Signal Transduction; Signaling Molecule; Testing; Translational Activation; Virulence; acute infection; cell motility; diguanylate cyclase; environmental change; enzyme activity; experience; genetic analysis; macrophage; metabolomics; mutant; novel; pathogen; pathogenic bacteria; phosphoric diester hydrolase

PROJECT SUMMARY Bacteria exist as single cells in a planktonic state, but they also associate into multicellular communities known as biofilms. A rise in the intracellular levels of the second messenger cyclic diguanylate (c-di-GMP) promotes biofilm formation in a wide range of bacterial species. Thus, understanding the control of c-di-GMP levels is essential to comprehend the transition between planktonic to biofilm states. Bacteria harbor diguanylate cyclases and phosphodiesterases, which are enzymes that synthesize and degrade c-di-GMP, respectively. A given bacterial species often harbors a large repertoire of these enzymes, each regulating a distinct cellular process. We reported that a Salmonella enterica serovar Typhimurium strain lacking the mgtC virulence gene harbors increased levels of c-di-GMP and of cellulose, a major component of biofilms that inhibits Salmonella replication inside macrophages. We have now identified the specific diguanylate cyclases responsible for the c-di-GMP- dependent cellulose synthesis in the mgtC mutant and established that they undergo post-translational activation. We have also utilized a novel c-di-GMP fluorescence reporter to select mutations (currently being mapped by high-throughput sequencing) that affect the activities of these diguanylate cyclases. This proposal seeks to define how the identified diguanylate cyclases are activated, and to determine the role of MgtC in controlling crucial physiological functions in Salmonella during infection. Specifically, the identification of genes controlling these diguanylate cyclases will enable us to explore how these enzymes are controlled during Salmonella replication inside macrophages and, by proxy, make testable inferences about metabolic changes promoted by MgtC. These investigations will reveal the cues promoting c-di-GMP synthesis and biofilm formation, and how bacterial pathogens must control their metabolism to replicate in host phagocytic cells.

项目概要 细菌以浮游状态的单细胞形式存在，但它们也结合成 称为生物膜的多细胞群落。细胞内的水平升高 第二信使环二鸟苷酸 (c-di-GMP) 广泛促进生物膜形成 细菌种类的范围。因此，了解 c-di-GMP 水平的控制是 对于理解浮游状态到生物膜状态之间的转变至关重要。细菌 含有二鸟苷酸环化酶和磷酸二酯酶，这些酶 分别合成和降解c-di-GMP。特定的细菌种类通常含有 这些酶的大量组成部分，每种酶都调节着不同的细胞过程。我们 报道称，肠沙门氏菌鼠伤寒血清型菌株缺乏 mgtC 毒力基因含有增加水平的 c-di-GMP 和纤维素，这是一种主要的 抑制巨噬细胞内沙门氏菌复制的生物膜成分。我们有 现在确定了负责 c-di-GMP- 的特定二鸟苷酸环化酶 mgtC 突变体中依赖的纤维素合成，并确定它们经历 翻译后激活。我们还利用了一种新型 c-di-GMP 荧光 记者选择突变（目前正在通过高通量测序进行定位） 影响这些二鸟苷酸环化酶的活性。该提案旨在定义 确定的二鸟苷酸环化酶是如何被激活的，并确定其作用 MgtC 在感染期间控制沙门氏菌的关键生理功能。 具体来说，控制这些二鸟苷酸环化酶的基因的鉴定将 使我们能够探索沙门氏菌复制过程中如何控制这些酶 在巨噬细胞内部，并通过代理对代谢变化做出可测试的推论 由 MgtC 推动。这些调查将揭示促进 c-di-GMP 的线索 合成和生物膜形成，以及细菌病原体必须如何控制它们 代谢在宿主吞噬细胞中复制。