Strategies for Acinetobacter baumannii to Maintain Zinc Homeostasis

鲍曼不动杆菌维持锌稳态的策略

• 批准号: 10667316
• 负责人: Jeanette Marie Miller
• 金额: $ 3.29万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667316
• 关键词: Acinetobacter baumannii; Affect; Anabolism; Bacteremia; Binding; Biochemical; Biological; Biological Assay; Cell Wall; Cells; Characteristics; Client; Data; Defect; Development; Diet; Dietary Zinc; Dietary intake; Ensure; Environment; Enzymes; Equilibrium; Generations; Genes; Genetic; Growth; Guanosine Triphosphate Phosphohydrolases; Homeostasis; Immune; Immune system; In Vitro; Infection; Innate Immune System; Iron; Laboratories; Leukocyte L1 Antigen Complex; Libraries; Life; Lytic; Maintenance; Malnutrition; Manganese; Mediating; Metalloproteins; Metals; Methods; Multi-Drug Resistance; Mus; Mutagenesis; Names; Nosocomial pneumonia; Nutrient; Nutritional Immunity; Organism; Pathogenesis; Pathway interactions; Peptidoglycan; Permeability; Phagocytes; Phenotype; Play; Pneumonia; Process; Property; Proteins; Reactive Oxygen Species; Recycling; Role; Sequence Homology; Series; Starvation; Stress; Testing; Therapeutic; Vertebrates; Virulence; Western Blotting; Work; Zinc; Zinc Chloride; Zinc deficiency; cell envelope; chelation; cofactor; combat; drug development; experience; experimental study; fitness; gene product; genome-wide; human pathogen; in vitro Assay; in vivo; insight; metal chelator; metalloenzyme; microbial; mouse model; mutant; new therapeutic target; novel therapeutics; opportunistic pathogen; pathogenic bacteria; pneumonia model; prevent; protein protein interaction; response; synergism; transposon sequencing

Project Summary All organisms must maintain a balance of nutrient metals to survive, including zinc (Zn). These metals are required as catalytic and structural cofactors for a variety of proteins, but in excess can lead to the generation of reactive oxygen species or inactivation of non-cognate enzymes through mismetallation. Therefore, tight control of metal levels through import, efflux, and storage is important for optimal growth and survival. Due to this requirement, bacterial metal homeostasis mechanisms are attractive targets for novel therapeutics. This proposal seeks to inform the development of metal-based therapies by identifying mechanisms used by the opportunistic pathogen Acinetobacter baumannii to prevent metal imbalance. Previous work in our laboratory has identified zigA which encodes a putative Zn metallochaperone with increased expression upon Zn starvation mediated by calprotectin, a metal sequestering protein of the innate immune system. Loss of ZigA results in a severe fitness defect upon calprotectin exposure, indicating the essentiality of ZigA under these conditions. Since ZigA is critical for A. baumannii to grow under Zn limitation, we hypothesized that proteins that receive metal from ZigA (clients) are equally important in mediating Zn stress. To identify these clients, I performed a genome- wide transposon mutagenesis screen in Zn limiting conditions comparing WT and ΔzigA libraries to identify genes whose fitness is influenced by Zn deficiency and that modulate the fitness of a ΔzigA mutant. I discovered several genes through this genetic interaction method and chose A1S_3027 for further characterization. A1S_3027 encodes a lytic transglycosylase that is predicted to tailor the bacterial cell wall. Strains lacking A1S_3027 are sensitized to Zn deficiency, and this can be reversed upon addition of ZnCl2. We hypothesize that to ensure the integrity of the cell envelope in conditions of Zn starvation, ZigA interacts with A1S_3027 to regulate its function. Characterizing A1S_3027 and its coordination with ZigA will be tested in two specific aims. In Specific Aim 1, I will study the biochemical properties and function of A. baumannii A1S_3027 using biochemical, genetic, and functional assays to probe how A1S_3027 helps to maintain Zn homeostasis. Experiments proposed in Specific Aim 2 will determine the functional role of A. baumannii A1S_3027 in maintaining appropriate nutrient metal balance by employing A1S_3027-deficient strains in a series of in vitro and in vivo experiments. Taken together, these aims will determine the impact of metal imbalance on A. baumannii pathogenesis and provide the first description of the contribution of a Zn metallochaperone and its clients to microbial virulence. Therapeutics that modulate bacterial metal levels will synergize with the immune system’s defenses, and A1S_3027 may be an attractive target for such metal-focused therapeutics.

项目摘要 所有生物必须保持营养金属的平衡才能生存，包括锌（Zn）。这些金属是 需要作为多种蛋白质的催化和结构辅助因子，但过量会导致产生 反应性氧或通过不易屈酶灭活非认知酶。因此，紧密控制 通过进口，外排和存储的金属水平对于最佳生长和生存至关重要。因此 要求，细菌金属体内稳态机制是新型治疗的有吸引力的靶标。这 提案旨在通过确定由 机会性病原体鲍曼尼（Baumannii）可防止金属失衡。我们实验室的先前工作 已经确定了编码假定的Zn金属伴侣的Ziga，在Zn饥饿时表达增加 由钙粘蛋白介导，钙骨蛋白是一种先天免疫系统的金属隔离蛋白。 Ziga的丢失导致 钙染色素暴露后严重的适应性缺陷，表明在这些条件下Ziga的本质。自从 Ziga对于A. baumannii在Zn限制下生长至关重要，我们假设接收金属的蛋白质 来自Ziga（客户）在介导Zn应力方面同样重要。为了识别这些客户，我进行了基因组 - 在Zn限制条件下，宽的转座子诱变屏幕比较WT和ΔZiga库以识别 适应性受Zn缺乏症影响并调节ΔZiga突变体的适应性的基因。我发现 通过这种遗传相互作用方法的几个基因，然后选择A1S_3027以进一步表征。 A1S_3027编码一个裂解的透明糖基化酶，该裂解糖基化酶预测可定制细菌细胞壁。缺乏菌株 A1S_3027对Zn缺乏症敏感，这可以在添加ZNCL2后反转。我们假设这一点 为了确保在Zn饥饿的条件下细胞信封的完整性，Ziga与A1S_3027相互作用以调节 它的功能。表征A1S_3027及其与Ziga的协调将以两个特定的目的进行测试。在 具体目标1，我将使用A. baumannii a1s_3027的生化特性和功能 生化，遗传和功能测定法，以探测A1S_3027如何有助于维持Zn稳态。 特定AIM 2中提出的实验将确定鲍曼尼A.3027在 通过在一系列体外使用A1S_3027缺乏菌株来维持适当的营养金属平衡 和体内实验。综上所述，这些目标将决定金属不平衡对A的影响。 Baumannii发病机理，并提供了锌金属伴侣及其贡献的首次描述 客户到微生物病毒。调节细菌金属水平的治疗剂将与免疫协同作用 系统的防御能力和A1S_3027可能是这种以金属为中心的治疗的有吸引力的目标。