Strategies for Acinetobacter baumannii to Maintain Zinc Homeostasis

鲍曼不动杆菌维持锌稳态的策略

基本信息

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
项目类别：
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

项目摘要

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.

项目概要所有生物体都必须维持营养金属的平衡才能生存，其中包括锌 (Zn)。需要作为多种蛋白质的催化和结构辅助因子，但过量会导致生成活性氧或通过金属错位使非同源酶失活，因此需要严格控制。因此，通过进口、流出和储存来控制金属水平对于最佳生长和生存非常重要。根据要求，细菌金属稳态机制是新型疗法的有吸引力的目标。该提案旨在通过确定金属疗法所使用的机制来为金属疗法的发展提供信息我们实验室以前的工作是利用机会性病原体鲍曼不动杆菌来防止金属失衡。已鉴定出 zigA，其编码假定的 Zn 金属伴侣，在 Zn 饥饿时表达增加由钙卫蛋白介导，钙卫蛋白是一种先天免疫系统的金属螯合蛋白，ZigA 的缺失会导致暴露于钙卫蛋白后出现严重的健康缺陷，表明 ZigA 在这些条件下的重要性。 ZigA 对于鲍曼不动杆菌在锌限制下生长至关重要，我们捕获了接受金属的蛋白质 ZigA（客户）在调节锌应激方面同样重要，为了识别这些客户，我进行了基因组分析。在 Zn 限制条件下进行宽转座子诱变筛选，比较 WT 和 ΔzigA 文库以鉴定我发现了一些基因，其适应性受到缺锌的影响，并调节 ΔzigA 突变体的适应性。通过这种遗传相互作用方法对几个基因进行了研究，并选择 A1S_3027 进行进一步表征。 A1S_3027 编码一种裂解性转糖基酶，预计可以调整缺乏细菌细胞壁的菌株。 A1S_3027 对缺锌敏感，添加 ZnCl2 可以逆转这种情况。为了确保锌饥饿条件下细胞膜的完整性，ZigA 与 A1S_3027 相互作用来调节 A1S_3027 的功能及其与 ZigA 的协调将在两个特定目标中进行测试。具体目标1，我将研究鲍曼不动杆菌A1S_3027的生化特性和功能生化、遗传和功能测定，以探讨 A1S_3027 如何帮助维持锌稳态。具体目标 2 中提出的实验将确定鲍曼不动杆菌 A1S_3027 在通过在一系列体外实验中使用 A1S_3027 缺陷菌株来维持适当的营养金属平衡综合起来，这些目标将确定金属失衡对 A 的影响。鲍曼不动杆菌的发病机制，并首次描述了锌金属伴侣及其贡献调节细菌金属水平的疗法将与免疫产生协同作用。系统的防御能力，A1S_3027 可能是此类金属聚焦疗法的一个有吸引力的目标。