Identification of a novel zinc acquisition system in Yersinia pestis

鼠疫耶尔森氏菌新型锌获取系统的鉴定

• 批准号: 10055730
• 负责人: Sarah Leann Price
• 金额: $ 3.26万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10055730
• 关键词: ATP-Binding Cassette Transporters; Antibiotic Resistance; Attenuated; Bacteria; Binding; Biochemical; Biological Assay; Bioterrorism; Bubonic Plague; Complement; Data; Defect; Disease; Environment; FDA approved; Gene Silencing; Genes; Gram-Negative Bacteria; Growth; Hemochromatosis; Human; In Vitro; Infection; Invaded; Iron; Leukocyte L1 Antigen Complex; Ligase; Manganese; Mediating; Metals; Methods; Modeling; Modernization; Modification; Mus; Nutrient; Nutritional Immunity; Organism; Pathogenesis; Phenotype; Plague; Play; Pneumonic Plague; Population; Production; Public Health; Role; Safety; Septicemic plague; Siderophores; Site; Societies; System; Techniques; Testing; Transition Elements; Vaccines; Virulence; Virulent; Yersinia pestis; Zinc; antimicrobial; defined contribution; experimental study; genetic element; in vivo; innovation; mouse model; mutant; neutrophil; new therapeutic target; novel; pandemic disease; pathogenic bacteria; targeted treatment; transposon sequencing; yersiniabactin; zinc-binding protein

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.

项目概要 鼠疫耶尔森氏菌是人类鼠疫的病原体。每年大约有 2,000 例人类鼠疫病例 过去曾发生过几次鼠疫大流行，造成了大范围的人口损失。鼠疫杆菌构成 由于它有可能被用作生物恐怖主义制剂，且缺乏 FDA 批准，因此对现代社会构成威胁 疫苗，以及抗生素耐药性的可能性。治疗药物新靶点的识别是 对公共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病原体。为了生存和 为了提高毒力，鼠疫耶尔森氏菌必须获得过渡金属，如铁、锌和锰，并克服营养免疫， 真核生物中限制营养物质入侵细菌的机制。人类瘟疫期间， 鼠疫耶尔森菌能够通过产生铁载体耶尔森菌素 (Ybt) 来克服铁限制。最近， 我们发现 Ybt 系统在鼠疫耶尔森氏菌感染过程中获取锌的能力中发挥着意想不到的作用。尽管 ZnuABC 系统有助于缺锌培养基中的体外生长，但 znu 突变体在 鼠疫小鼠模型，除非突变体也缺乏参与 Ybt 合成的基因。这些数据表明 鼠疫耶尔森氏菌使用两个冗余的锌获取系统来引起鼠疫。我们假设鼠疫耶尔森氏菌产生 锌获取和毒力所需的新型 Ybt 合成酶依赖性锌载体。在具体目标 1 中，我们 将使用一种新颖的 Tn-seq 方法来定义锌载体系统中涉及的遗传元件。显示的基因 锌表型将通过生长测定、反式互补和生化实验进行验证。 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 利用血色素沉着病小鼠模型来确定毒力。血色病小鼠存在铁缺陷 营养免疫，这将使我们能够区分 Ybt 合成酶依赖性 Fe 的贡献 鼠疫耶尔森氏菌毒力的获取和 Ybt 合成酶依赖性 Zn 获取。在具体目标 3 中，我们将 使用体外和体内方法确定钙卫蛋白对鼠疫耶尔森菌毒力的影响。完成 这些目标将定义鼠疫耶尔森氏菌中新型分泌型锌获取系统。我们的研究将首先确定 锌获取对哺乳动物宿主鼠疫杆菌毒力的贡献以及钙卫蛋白的影响 鼠疫感染。此外，保守的 Ybt 参与这种新颖的 Zn 收购表明 这些研究对其他细菌病原体的重要性。