Identification of a novel zinc acquisition system in Yersinia pestis

项目摘要 鼠疫耶尔森氏菌是人类鼠疫的病原体。每年大约有2,000例人间鼠疫 在过去，几次鼠疫大流行造成了广泛的人口损失。Y.鼠疫姿势 由于其可能被用作生物恐怖主义制剂，因此对现代社会构成威胁， 疫苗和抗生素耐药性的可能性。治疗剂的新靶点的鉴定是 对公众健康和安全至关重要。因此，Y.鼠疫被认为是一级选择代理。为生存和 毒力、Y.鼠疫必须获得过渡金属，如铁，锌，锰和克服营养免疫， 真核生物中限制营养素入侵细菌的机制。在人类瘟疫期间， 鼠疫耶尔森氏菌能够通过产生铁载体耶尔森氏菌素（Ybt）来克服铁限制。最近， 我们确定了Ybt系统在Y.鼠疫菌在感染过程中获得锌。而 ZnuABC系统有助于在缺锌培养基中的体外生长，znu突变体在培养基中不减毒。 鼠疫的小鼠模型，除非突变体也缺乏参与Ybt合成的基因。这些数据表明 Y.鼠疫菌使用两个冗余的锌获取系统来引起鼠疫。我们假设Y.鼠疫产生 一种新的依赖Ybt合成酶的锌载体，锌的获得和毒力所需。在具体目标1中， 将使用一种新的Tn-seq方法来定义锌载体系统中涉及的遗传元件。基因显示 a Zn表型将通过生长测定、反式互补和生物化学实验来验证。 在具体目标2中，我们将确定Ybt合成酶依赖性锌获取系统对 利用血色素沉着症小鼠模型，血色素沉着症小鼠缺铁 营养免疫，这将使我们能够区分Ybt合成酶依赖的Fe 获得和Ybt合成酶依赖的Zn获得。鼠疫毒力在具体目标3中，我们 确定钙卫蛋白对Y.鼠疫菌毒力的研究。完成 这些目的将在Y.鼠疫我们的研究将首先确定 Zn的吸收对Y.鼠疫菌在哺乳动物宿主中的毒力以及钙卫蛋白对 鼠疫感染此外，保守的Ybt参与这种新的Zn获得表明， 这些研究对其他细菌病原体的意义。

PROJECT SUMMARY Yersinia pestis is the causative agent of human plague. Every year, approximately 2,000 cases of human plague occur, and in the past, several pandemics of plague have caused wide spread population loss. Y. pestis poses a threat to modern society due to its potential to be used as a bioterrorism agent, the absence of a FDA approved vaccine, and the possibility of antibiotic resistance. The identification of novel targets for therapeutic agents is critical for public health and safety. Consequently, Y. pestis is considered a Tier 1 Select Agent. For survival and virulence, Y. pestis must acquire transition metals, such as Fe, Zn, and Mn and overcome nutritional immunity, mechanisms in eukaryotic organisms that restrict nutrients from invading bacteria. During human plague, Yersinia pestis is able to overcome Fe limitation via production of the siderophore Yersiniabactin (Ybt). Recently, we identified an unexpected role for the Ybt system in the ability of Y. pestis to acquire Zn during infection. While the ZnuABC system contributes to in vitro growth in Zn-deficient media, a znu mutant is not attenuated in the mouse model of plague, unless the mutant also lacks genes involved in Ybt synthesis. These data suggest that Y. pestis uses two redundant Zn acquisition systems to cause plague. We hypothesize that Y. pestis produces a novel Ybt synthetase-dependent zincophore required for zinc acquisition and virulence. In Specific Aim 1, we will use a novel Tn-seq method to define genetic elements involved in the zincophore system. Genes that show a Zn phenotype will be validated through growth assays, trans-complementation, and biochemical experiments. In Specific Aim 2, we will determine the contribution of the Ybt synthetase-dependent zinc acquisition system to virulence by utilizing a hemochromatosis mouse model. The hemochromatosis mouse is defective in Fe nutritional immunity, which will allow us to distinguish between the contributions of Ybt synthetase-dependent Fe acquisition and Ybt synthetase-dependent Zn acquisition to Y. pestis virulence. In Specific Aim 3, we will determine the impact of calprotectin on Y. pestis virulence by using in vitro and in vivo methods. Completion of these Aims will define a novel secreted Zn acquisition system in Y. pestis. Our studies will be the first to determine the contribution of Zn acquisition to Y. pestis virulence in the mammalian host and the effect of calprotectin on plague infection. Furthermore, the involvement of conserved Ybt in this novel Zn acquisition demonstrates the significance of these studies to other bacterial pathogens.