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: 10990786
• 负责人: Zaara Sarwar
• 金额: $ 35.51万
• 依托单位: EASTERN WASHINGTON UNIVERSITY
• 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 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10990786
• 关键词: role; master; regulator; NtrC; amyloid

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种多样的病原体，能够感染多种人体组织并造成 不可逆转的、危及生命的损害。铜绿假单胞菌不仅可以依靠从细菌中释放的营养物质生存 破坏宿主细胞和/或组织，但重要的是，这种细菌有适应和保护自己的倾向 免受对其采取的任何防御措施，无论是来自寄主有机体的免疫反应还是 外部补救措施，如抗生素治疗。拟议项目的长期目标是中和或 通过特异性靶向干扰铜绿假单胞菌致病机制来对抗这些关键方面 增强子结合蛋白-RpoN调控网络。RpoN和EBPS相互作用以激活 &gt；200靶基因在铜绿假单胞菌中的转录。许多以EBP-RpoN为靶标的基因对于 利用寄主来源的营养物质，并提供对包括过氧化氢在内的细胞损害剂的抵抗力， 高温和抗生素。该提案的中心假设是，EBP NTRC调节 淀粉样蛋白纤维−以及允许铜绿假单胞菌与真核细胞结合并获得 在宿主环境中站稳脚跟。这项拟议研究的基本原理是，对基因- NTRC的调控网络和致病作用将提供对分子机制和 协调铜绿假单胞菌适应真核宿主环境的信号。在具体目标1中， 将定义NTRC的规则。NTRC的染色体DNA结合部位将通过染色质确定 核酸外切酶免疫沉淀高通量测序(CHIP-EXO)。此外， NTRC的转录组将使用RNA-seq进行鉴定。芯片-exo和rna-seq的结果将结合在一起 确定由NTRC直接管理的基因调控网络。在特定的目标2中，淀粉样蛋白纤维和 它的调节剂NTRC将被研究对真核细胞的黏附和细胞毒性。这个 方法是创新的，因为它将确定细胞是否需要淀粉样纤维及其调节因子NTRC 铜绿假单胞菌在宿主环境中的粘附性。它还将用作在 其他病原微生物中EBPs或DNA结合蛋白的特征。我们越来越接近 对EBP-RpoN在这种重要的人类病原体中的调控有一个完整的定义。拟议中的项目 具有重要意义，因为了解这一调控及其控制的抗性机制将有助于 发展抗菌剂和替代方法来对抗P。 铜绿假单胞菌。