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

项目摘要 细菌以单细胞的形式存在，但它们也能结合成 称为生物膜的多细胞群落。在细胞内水平的上升， 第二信使环二鸟苷酸（c-di-GMP）促进生物膜形成，在广泛的 细菌种类的范围。因此，了解c-di-GMP水平的控制是 这对于理解生物膜状态与非生物张力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。一个特定的细菌种类通常含有 这些酶的一个大的库，每一个调节一个不同的细胞过程。我们 报道了缺乏mgtC的肠道沙门氏菌血清型鼠伤寒沙门氏菌菌株， 毒力基因窝藏增加水平的c-di-GMP和纤维素，一个主要 抑制沙门氏菌在巨噬细胞内复制的生物膜成分。我们有 现在确定了负责c-di-GMP的特异性二鸟苷酸环化酶， 依赖于mgtC突变体中的纤维素合成，并确定它们经历 翻译后激活。我们还利用了新的c-di-GMP荧光 选择突变的报告基因（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。该提案旨在界定 如何识别的二鸟苷酸环化酶被激活，并确定的作用， MgtC在沙门氏菌感染过程中控制重要的生理功能。 具体而言，控制这些二鸟苷酸环化酶的基因的鉴定将 使我们能够探索在沙门氏菌复制过程中这些酶是如何被控制的 在巨噬细胞内，通过代理，对代谢变化做出可测试的推断， 由MgtC推广。这些研究将揭示促进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的 代谢在宿主吞噬细胞中复制。

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.