Identification of a novel zinc acquisition system in Yersinia pestis

项目总结 鼠疫耶尔森氏菌是人类鼠疫的病原体。每年，大约有2000例人类瘟疫 在过去的几次鼠疫大流行中，曾造成大范围的人口损失。鼠疫耶尔森氏菌构成 由于其可能被用作生物恐怖主义毒剂而对现代社会构成威胁，没有FDA批准的 疫苗，以及抗生素抗药性的可能性。治疗药物的新靶点的鉴定是 对公众健康和安全至关重要。因此，鼠疫耶尔森氏菌被认为是一级选择剂。为了生存和 毒力，鼠疫杆菌必须获得过渡金属，如铁，锌和锰，并克服营养免疫， 真核生物中限制来自入侵细菌的营养的机制。在人类瘟疫期间， 鼠疫耶尔森菌能够通过产生铁载体耶尔森纳巴菌素(Ybt)来克服铁的限制。最近， 我们发现Ybt系统在鼠疫杆菌感染期间获得锌的能力中发挥了意想不到的作用。而当 ZnuABC系统有助于在缺锌介质中的体外生长，ZNU突变体在 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 鼠疫杆菌使用两个多余的锌获取系统来引起鼠疫。我们假设鼠疫杆菌产生 一种新的依赖于Ybt合成酶的锌载体，需要锌的获取和毒力。在具体目标1中，我们 将使用一种新的TN-SEQ方法来定义Zincohore系统中涉及的遗传元素。基因显示出 锌的表型将通过生长分析、反式互补和生化实验来验证。 在特定的目标2中，我们将确定依赖于Ybt合成酶的锌获取系统对 利用血色素沉着症小鼠模型进行毒力测定。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的贡献 鼠疫耶尔森氏菌毒力的获得和Ybt合成酶依赖的锌获得。在具体目标3中，我们将 用体外和体内方法测定钙保护素对鼠疫杆菌毒力的影响。完成 这些目的将定义一种新的鼠疫菌分泌型锌获取系统。我们的研究将首先确定 在哺乳动物宿主中获取锌对鼠疫杆菌毒力的贡献以及钙保护素对其毒力的影响 鼠疫感染。此外，保守的Ybt参与了这一新的锌获取，证明了 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.