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.

项目摘要/摘要 铜绿假单胞菌是一种多才多艺的病原体，能够感染众多人体组织并造成 不可逆的威胁生命的损害。铜绿假单胞菌不仅可以在从 受损的宿主细胞和/或组织，但重要的是，该细菌有望适应和保护自身 无论是根据寄主生物的免疫反应还是采取任何防御措施 外部疗法，例如抗生素治疗。这是拟议项目的长期目标，即中和或 通过特异性靶向破坏来对抗铜绿假单胞菌发病机理的这些关键方面 增强子结合蛋白（EBP） - RPON调节网络。 RPON和EBP相互交互以激活 铜绿假单胞菌中> 200个靶基因的转录。许多EBP-RPON靶向基因对于 利用宿主衍生的养分，并提供对细胞破坏剂的抗性，包括过氧化物， 热和抗生素。该提案的中心假设是EBP NTRC调节的表达 淀粉样蛋白纤维 - 以及其他蛋白质 - 允许铜绿假单胞菌与真核细胞结合并获得 主机环境中的立足点。拟议的研究的理由是，对基因的了解 NTRC的调节网络和致病作用将为分子机制和 协调铜绿假单胞菌对真核生物宿主环境的适应的信号。在特定的目标1中 NTRC的法规将定义。 NTRC的染色体DNA结合位点将通过染色质确定 外丝酶治疗高通量测序（CHIP-EXO）的免疫沉淀。另外， NTRC的转录组将使用RNA-seq确定。芯片-EXO和RNA-seq的结果将 确定直接由NTRC控制的基因调节网络。在特定的目标2中，淀粉样纤维原纤维的作用和 它的调节剂NTRC将根据对真核细胞的粘附和细胞毒性进行研究。这 方法是创新的，因为它将确定淀粉样蛋白纤维及其调节剂NTRC是否需要细胞 铜绿假单胞菌在宿主环境中的粘附。它还将用作使用芯片exo的模板 其他致病微生物中EBP或DNA结合蛋白的表征。我们越来越近 在这种重要的人类病原体中对EBP-RPON调节具有完整的定义。拟议的项目 很重要，因为对此法规的知识及其控制的抵抗机制将有助于 抗菌剂和替代方法的开发，以应对P的病毒或发病机理。 铜绿。