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 所有生物都必须保持营养金属的平衡才能生存，包括锌（Zn）。这些金属 作为多种蛋白质的催化和结构辅因子所需，但过量可导致 活性氧物质或通过错代谢使非同源酶失活。因此，严格控制 通过输入，流出和储存的金属水平对于最佳生长和存活是重要的。由于这种 根据这一要求，细菌金属稳态机制是新疗法的有吸引力的靶标。这 该提案旨在通过确定金属基疗法所使用的机制来为金属基疗法的发展提供信息。 条件致病菌鲍曼不动杆菌，以防止金属失衡。我们实验室以前的工作 已经鉴定了zigA，其编码假定的锌金属伴侣蛋白，在锌饥饿时表达增加 由钙卫蛋白介导，钙卫蛋白是先天免疫系统的金属螯合蛋白。ZigA的缺失导致 严重的健身缺陷后，钙卫蛋白暴露，表明ZigA在这些条件下的必要性。以来 ZigA是A的关键。鲍曼不动杆菌在锌限制下生长，我们假设接受金属的蛋白质 来自ZigA（客户）的锌在介导锌应激中同样重要。为了确认这些客户，我做了一个基因组- 在Zn限制条件下比较WT和ΔzigA文库进行宽转座子诱变筛选，以鉴定 适应性受锌缺乏影响并调节ΔzigA突变体适应性的基因。我发现 通过这种遗传互作方法筛选了几个基因，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，预测该酶可调节细菌细胞壁。缺乏菌株 A1S_3027对Zn缺乏敏感，并且这可以在添加ZnCl 2时逆转。我们假设 为了确保锌饥饿条件下细胞被膜的完整性，ZigA与A1S_3027相互作用， 它的功能。A1S_3027的特性及其与ZigA的协调性将在两个特定目标中进行测试。在 具体目的1、研究A.鲍曼不动杆菌A1S_3027， 生物化学、遗传和功能测定来探测A1S_3027如何帮助维持Zn稳态。 具体目标2中提出的实验将确定A的功能作用。鲍曼不动杆菌A1S_3027在 通过在一系列体外试验中使用A1S_3027缺陷型菌株来维持适当的营养金属平衡 和体内实验。综合考虑，这些目标将决定金属不平衡对A的影响。 鲍曼不动杆菌的发病机制，并提供了锌金属伴侣的贡献和它的第一个描述。 微生物毒性的客户。调节细菌金属水平的治疗剂将与免疫系统协同作用。 A1S_3027可能是这种金属聚焦疗法的一个有吸引力的靶点。