The role of master regulator NtrC in amyloid fibril dependent pathogenic traits of Pseudomonas aeruginosa

主调节因子 NtrC 在铜绿假单胞菌淀粉样原纤维依赖性致病性状中的作用

• 批准号: 10652868
• 负责人: Zaara Sarwar
• 金额: $ 2.8万
• 依托单位: COLLEGE OF NEW JERSEY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10652868
• 关键词: Adhesions; Affect; Amyloid Fibrils; Animal Model; Antibiotic Therapy; Antibiotics; Bacteria; Behavior; Binding; Binding Proteins; Binding Sites; Biological; Cell Adhesion; Cell Adhesion Molecules; Cells; Cues; DNA Binding; DNA-Binding Proteins; Development; Environment; Epithelial Cells; Eukaryotic Cell; Exonuclease; Exposure to; Family; Future; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Goals; Health; High-Throughput Nucleotide Sequencing; Human; Immune response; Infection; Investigation; Iron; Knowledge; Life; Measures; Methodology; Microbial Biofilms; Mission; Molecular; Nature; Nitrogen; Nutrient; Organism; Outcome; Pathogenesis; Pathogenicity; Peroxides; Positioning Attribute; Process; Production; Proteins; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Public Health; Regulation; Regulon; Research Personnel; Resistance; Role; Sigma Factor; Signal Transduction; Tissues; United States National Institutes of Health; Virulence; Virulence Factors; airway epithelium; antimicrobial; burden of illness; cell injury; chromatin immunoprecipitation; combat; cytotoxicity; enhancer binding protein; enteric pathogen; extracellular; gene regulatory network; genetic regulatory protein; human pathogen; human tissue; innovation; insight; microbial; microorganism; mutant; pathogen; prevent; resistance mechanism; trait; transcriptome; transcriptome sequencing

Project Summary/Abstract Pseudomonas aeruginosa is a versatile pathogen, capable of infecting numerous human tissues and inflicting irreversible, life-threatening damage. Not only can P. aeruginosa survive on the nutrients released from the damaged host cells and/or tissues, but importantly, this bacterium has the propensity to adapt and protect itself from any defensive measures taken against it whether from the immune response of the host organism or external remedies such as antibiotic treatments. It is the long-term goal of the proposed project to neutralize or counter these key aspects of P. aeruginosa pathogenesis through the specific targeted disruption of the enhancer-binding protein (EBP)-RpoN regulatory network. RpoN and EBPs interact with each other to activate transcription of >200 target genes in P. aeruginosa. Many of the EBP-RpoN targeted genes are essential for the utilization of host-derived nutrients and provide resistance against cell-damaging agents, including peroxides, heat and antibiotics. The central hypothesis of the proposal is that the EBP NtrC regulates the expression of amyloid fibrils − as well as additional proteins − that allow for P. aeruginosa to bind to eukaryotic cells and gain a foothold in the host environment. The rationale for the proposed study is that knowledge of both the gene- regulatory networks and pathogenic roles for NtrC will provide crucial insight into the molecular mechanisms and signals that coordinate the adaptation of P. aeruginosa to a eukaryotic-host environment. In Specific Aim 1, the regulon of NtrC will be defined. The chromosomal DNA-binding sites for NtrC will be determined via chromatin immunoprecipitation with exonuclease treatment-high-throughput sequencing (ChIP-exo). Additionally, the transcriptome of NtrC will be identified using RNA-seq. The results of the ChIP-exo and RNA-seq together will identify the gene-regulatory network directly governed by NtrC. In Specific Aim 2, the roles of amyloid fibrils and its regulator NtrC will be investigated in terms of adhesion and cytotoxicity towards eukaryotic cells. The approach is innovative because it will establish whether amyloid fibrils and its regulator NtrC are required for cell adhesion of P. aeruginosa in a host environment. It will also serve as a template for using ChIP-exo in the characterization of EBPs or DNA-binding proteins in other pathogenic microorganisms. We are getting closer to having a complete definition of EBP-RpoN regulation in this important human pathogen. The proposed project is significant, because knowledge of this regulation and the resistance mechanisms it controls will facilitate the development of antimicrobials and alternative approaches to counter the virulence or pathogenesis of P. aeruginosa.

项目概要/摘要 铜绿假单胞菌是一种多功能病原体，能够感染多种人体组织并造成 不可逆的、危及生命的损害。铜绿假单胞菌不仅可以依靠释放的营养物质生存。 损坏宿主细胞和/或组织，但重要的是，这种细菌具有适应和保护自身的倾向 对其采取的任何防御措施，无论是宿主生物体的免疫反应还是 拟议项目的长期目标是消除或消除抗生素治疗等外部疗法。 通过特定目标破坏铜绿假单胞菌发病机制的这些关键方面 增强子结合蛋白（EBP）-RpoN 调节网络和 RpoN 相互作用以激活。 铜绿假单胞菌中超过 200 个靶基因的转录 许多 EBP-RpoN 靶基因对于铜绿假单胞菌中的转录至关重要。 利用宿主来源的营养物质并提供对细胞损伤剂（包括过氧化物）的抵抗力， 该提案的中心假设是 EBP NtrC 调节 的表达。 淀粉样原纤维以及其他蛋白质，使铜绿假单胞菌能够与真核细胞结合并获得 拟议研究的基本原理是对基因的了解。 NtrC 的调控网络和致病作用将为分子机制和 在特定目标 1 中，协调铜绿假单胞菌适应真核宿主环境的信号。 NtrC 的调节子将被定义 NtrC 的染色体 DNA 结合位点将通过染色质确定。 核酸外切酶处理免疫沉淀-高通量测序 (ChIP-exo)。 NtrC 的转录组将使用 RNA-seq 进行鉴定 ChIP-exo 和 RNA-seq 的结果将一起进行。 确定由 NtrC 直接控制的基因调控网络，在具体目标 2 中，淀粉样原纤维和淀粉样蛋白纤维的作用。 其调节因子 NtrC 将在对真核细胞的粘附和细胞毒性方面进行研究。 该方法具有创新性，因为它将确定细胞是否需要淀粉样原纤维及其调节因子 NtrC 它也将作为在宿主环境中使用 ChIP-exo 的模板。 我们正在进一步了解其他病原微生物中 EBP 或 DNA 结合蛋白的特征。 对这种重要的人类病原体的 EBP-RpoN 调节有完整的定义。 意义重大，因为了解这一调节及其控制的抵抗机制将有助于 开发抗微生物药物和替代方法来对抗疟原虫的毒力或发病机制。 铜绿假单胞菌。