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

项目总结 细菌以浮游状态的单个细胞存在，但它们也与 被称为生物膜的多细胞群落。细胞内细胞内水平的上升 第二信使环二鸟苷(c-di-GMP)促进生物被膜的形成 细菌种类的范围。因此，理解c-di-GMP水平的控制是 对于理解从浮游状态到生物膜状态的转变至关重要。细菌 港湾二鸟苷环化酶和磷酸二酯酶，这是一种酶 分别合成和降解c-di-GMP。一种给定的细菌物种通常含有 这些酶的一大套，每一种都调节一个不同的细胞过程。我们 报道一株缺乏MGTC的肠出血性沙门氏菌 毒力基因含有c-di-GMP和纤维素的水平增加，纤维素是一种主要的 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的特定的二鸟苷晚期环化酶- MGTC突变体中依赖的纤维素合成，并证实它们经历了 翻译后激活。我们还利用了一种新的c-di-GMP荧光 选择突变的记者(目前正在通过高通量测序绘制图谱) 会影响这些二鸟苷晚期环化酶的活性。这项提案旨在界定 如何激活已识别的二鸟苷酸环化酶，并确定其作用 MGTC在控制沙门氏菌感染过程中的关键生理功能。 具体地说，控制这些二鸟苷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控制的 在巨噬细胞内，并通过代理对代谢变化做出可测试的推断 由MGTC推动。这些调查将揭示促进c-di-GMP的线索 合成和生物被膜的形成，以及细菌病原体必须如何控制其 在宿主吞噬细胞中进行复制的代谢。

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.