A cyclic di-GMP signaling system of spores of Bacillus anthracis

 描述(由申请人提供)：炭疽芽孢杆菌是一种形成内孢子的人畜共患病细菌，作为生物恐怖主义因子而备受关注。孢子进入人类宿主的途径决定了由此产生的疾病的性质，是皮肤病、胃肠道病还是肺病。胃肠道和肺部炭疽病与高死亡率有关。炭疽杆菌孢子的组成，特别是其间隙层和外孢子层的组成尚不完全清楚。酶，包括丙氨酸消旋酶、肌苷偏爱核苷水解酶和一氧化氮合酶，存在于外孢子层、间隙和外孢子层。这些酶被认为在孢子萌发和对宿主天然免疫系统的防御中发挥作用。我们已经确定在炭疽杆菌孢子中存在与调节环状双GMP水平相关的蛋白质。环二核苷酸是一种重要的调节因子，已在许多细菌病原体中被发现。它是许多毒力属性的关键调节因子，包括生物膜的形成、运动性、细胞内存活、表面黏附的表达、菌毛的表达、对氧化应激的抵抗以及宿主免疫反应的调节。这种环核苷酸调节因子在孢子生物学中的作用，无论是孢子形成还是萌发，都没有报道。我们建议研究含有GGDEF结构域的蛋白，其表达谱和/或蛋白融合研究表明它们是孢子蛋白。对这些决定因素之一的初步研究表明，对孢子成熟有影响。在这个项目中，我们将确定这些GGDEF蛋白在孢子中的位置，这些蛋白单独和联合丢失对孢子结构和功能的影响，以及对巨噬细胞培养的孢子生物学和感染小鼠模型的影响。这些实验的目的是确定GGDEF蛋白在产孢量、萌发和毒力上的作用。由于孢子是这种人畜共患病病原体的感染形式，这一信息对我们了解炭疽杆菌的感染过程很重要，并可能确定针对炭疽杆菌感染的最初步骤进行治疗干预的潜在靶点。这项研究获得的信息可能对了解其他内孢子形成细菌病原体的感染过程具有更广泛的适用性。

 DESCRIPTION (provided by applicant): Bacillus anthracis is an endospore-forming zoonotic bacterium of significant concern as a bioterrorism agent. The route by which the spore gains access to a human host defines the nature of the resulting disease, cutaneous, gastrointestinal, or pulmonary. Gastrointestinal and pulmonary anthrax are associated with high mortality rates. The composition of the B. anthracis spore, especially its interspace and exosporium layers, is incompletely understood. Enzymes, including alanine racemase, inosine preferring nucleoside hydrolase, and nitric oxide synthase, are found in the outer spore coat, interspace, and exosporium layers. These enzymes are thought to function in spore germination and defense against the host innate immune system. We have identified the presence of proteins associated with the regulation of cyclic di-GMP levels in spores of B. anthracis. Cyclic di-GMP is an important regulator that has been identified in a number of bacterial pathogens. It is a critical regulator of a number of virulence attributes including biofilm formation, motility, intracellular survival, expression of surface adhesions, expression of pili, resistance to oxidative stress, and modulation of the host immune response. A role for this cyclic nucleotide regulator in spore biology, either sporulation or germination, has not been reported. We propose to study GGDEF domain-containing proteins whose expression profile and/or protein fusion studies indicate that they are spore proteins. Initial studies with one of these determinants have shown an effect on spore maturation. In this project, we will determine the location within the spores for these GGDEF proteins, the effects on spore structure and function associated with loss of these proteins both singly and in combination, and the effects on spore biology in macrophage cell cultures and a mouse model of infection. The goals of these experiments are to determine the roles of the GGDEF proteins on sporulation, germination, and virulence. Because the spore is the infectious form of this zoonotic pathogen, this information is important to our understanding of the B. anthracis infectious process and may identify potential targets for therapeutic intervention against the initial steps in B. anthracis infections. Information obtained from this study may have broader applicability to understanding the infectious process of other endospore-forming bacterial pathogens.