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.

项目总结/摘要 铜绿假单胞菌是一种多功能病原体，能够感染许多人体组织并造成 不可逆转的危及生命的损伤铜绿假单胞菌不仅可以依靠从细菌中释放的营养物质存活， 受损的宿主细胞和/或组织，但重要的是，这种细菌有适应和保护自己的倾向 从任何防御措施，无论是从宿主生物体的免疫反应， 外部治疗，如抗生素治疗。拟议项目的长期目标是中和或 通过特异性靶向破坏铜绿假单胞菌致病机制的这些关键方面， 增强子结合蛋白（EBP）-RpoN调控网络。RpoN和EBP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靶向基因对于细胞的增殖和分化是必需的。 利用宿主来源的营养素并提供对细胞损伤剂的抗性，包括过氧化物， 加热和抗生素。该提案的中心假设是EBP NtrC调节以下表达： 淀粉样纤维-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 在宿主环境中的立足点这项研究的基本原理是，了解基因- NtrC的调节网络和致病作用将为分子机制提供重要见解， 协调铜绿假单胞菌适应真核宿主环境的信号。具体目标1： 将定义NtrC的调节子。NtrC的染色体DNA结合位点将通过染色质 用核酸外切酶处理免疫沉淀-高通量测序（ChIP-exo）。另夕h 将使用RNA-seq鉴定NtrC的转录组。ChIP-exo和RNA-seq的结果将 确定由NtrC直接管理的基因调控网络。在特异性目标2中，淀粉样纤维和 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。的 这种方法是创新的，因为它将确定淀粉样纤维及其调节因子NtrC是否是细胞增殖所必需的。 铜绿假单胞菌在宿主环境中的粘附。它还将作为在生物技术中使用ChIP-exo的模板。 EBP或DNA结合蛋白在其他病原微生物的表征。我们越来越接近 在这种重要的人类病原体中具有EBP-RpoN调节的完整定义。拟建项目 是重要的，因为了解这种调节及其控制的抗性机制将有助于 开发抗微生物剂和替代方法来对抗P. 铜绿。