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 所有生物体都必须保持营养金属的平衡才能生存，包括锌。这些金属是 作为各种蛋白质的催化和结构辅助因子所需，但过量会导致产生 非同系酶的活性氧种或失活，通过混合金属作用。因此，严格控制 通过进口、流出和储存来控制金属水平对于最佳生长和存活很重要。由于这一点 要求，细菌金属动态平衡机制是新的治疗药物吸引人的靶点。这 一项提案旨在通过确定金属疗法使用的机制来为基于金属的疗法的发展提供信息 机会性致病菌鲍曼不动杆菌预防金属失衡。我们实验室以前的工作 已经发现ZIGA编码了一种可能的锌金属配位体，在锌饥饿时表达增加 由钙保护素介导，这是一种天然免疫系统的金属隔离蛋白。失去ZIGA导致 钙保护素暴露后的严重体能缺陷，说明了ZIGA在这种条件下的必要性。自.以来 ZIGA是鲍曼曲霉在锌限制下生长的关键，我们假设接受金属的蛋白质 ZIGA(客户)在调节锌胁迫方面也同样重要。为了辨认这些客户，我进行了基因组测试- 锌限制条件下广谱转座子诱变WT和ΔZIGA文库的比较研究 其适合性受缺锌影响的基因，以及调节ΔZIGA突变体适合性的基因。我发现 通过这种遗传互作方法，选择了A1S_3027进行进一步的鉴定。 A1S_3027编码一种裂解转糖基酶，预计该酶可定制细菌细胞壁。缺少菌株 AlS_3027对缺锌有敏化作用，加入ZnCl2可逆转缺锌。我们假设 为了确保锌饥饿条件下细胞膜的完整性，ZIGA与A1S_3027相互作用调节 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下进行测试。在……里面 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、遗传和功能分析以探讨A1S_3027如何帮助维持锌的动态平衡。 在特定目标2中提出的实验将确定鲍曼不动杆菌A1S_3027在 利用A1S_3027缺陷菌株在一系列体外培养中维持适当的营养金属平衡 以及活体实验。综上所述，这些目标将决定金属失衡对A。 鲍曼氏杆菌的致病机制，并首次描述了锌金属配位酮及其作用。 客户受到微生物毒力的影响。调节细菌金属水平的治疗药物将与免疫协同作用 A1S_3027可能是这类以金属为重点的疗法的一个有吸引力的靶点。