Systematic analysis of Proteus mirabilis transcriptional regulators that drive uropathogenesis

驱动尿路病理发生的奇异变形杆菌转录调节因子的系统分析

• 批准号: 10594401
• 负责人: Madison Jane Fitzgerald
• 金额: $ 4.13万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594401
• 关键词: Accounting; Adherence; Agar; Antibiotic Resistance; Bacteria; Biochemical; Bioinformatics; Biological Assay; Biology; Bladder; Bladder Calculi; Catheters; Cell physiology; Complement; Cues; Data Set; Development; Environment; Essential Genes; Flagella; Future; Genes; Genetic Transcription; Genome; Goals; Growth; Harvest; Healthcare Systems; Hour; In Vitro; Individual; Infection; Iron; Knowledge; Learning; Libraries; Literature; Maintenance; Mediating; Methodology; Methods; Microbial Biofilms; Molecular; Mus; Mutagenesis; Names; Natural Resistance; Nosocomial Infections; Nutrient; Organism; Outcome; Output; Pathogenesis; Patients; Phenotype; Physicians; Plasmids; Polymyxins; Production; Proteus mirabilis; Publishing; Quantitative Reverse Transcriptase PCR; Regulon; Source; System; Techniques; Testing; Tetracycline Resistance; Therapeutic; Toxin; United States; Urease; Urinary Calculi; Urinary tract; Urinary tract infection; Urination; Urine; Vaccines; Virulence; Virulence Factors; Visit; bioinformatics tool; catheter associated UTI; comparative; cost; experimental study; fitness; follow-up; genetic approach; genome-wide; in vivo; inducible gene expression; interest; knowledge base; member; mouse model; mutant; novel; novel therapeutics; pathogen; promoter; response; screening; targeted treatment; transcription factor; transcriptome sequencing; transcriptomics; transposon sequencing; uptake; vector

ABSTRACT Urinary Tract Infections (UTIs) are common infections that represent a significant burden on healthcare systems worldwide. In 2006 alone, UTIs were the source of 11 million physician visits and cost the United States over $3.5 billion. A significant portion of these infections are Catheter-Associated UTIs (CAUTIs), accounting for up to 40% of hospital acquired infections globally. A major cause of CAUTIs is Proteus mirabilis, an understudied Gram-negative member of the Enterobacterales order. This organism is most noted for its ability to swarm on agar and form urinary stones in infected patients. Previous studies have used a mouse model of UTI to identify factors that contribute to fitness and virulence in the urinary tract. While these datasets strongly implicate transcriptional regulators, there is much to learn about transcriptional networks in this species. Toward this end, I have developed the framework to employ Transcriptional Regulator Induced Phenotype (TRIP) screening to identify specific regulators that contribute to uropathogenesis. TRIP leverages a library of strains containing inducible expression constructs that each encode a single regulator. Pools of these strains will be inoculated into the mouse model of UTI to assess comparative fitness. Sequencing and bioinformatic analyses will be used to assess relative fitness of TRIP strains and identify strains with a competitive edge in the bladder environment. After identifying these key regulators, I aim to define the downstream regulatory networks using RNA-sequencing and employ genetic approaches, biochemical assays, and the murine model of UTI to ascertain the molecular mechanisms behind the fitness advantage. Using a systematic bioinformatics approach, I have identified 232 putative transcriptional regulators in the HI4320 genome. Only 3 of these regulators have defined regulons published in the literature. Thus far, 35/232 (15%) of the constructs have been generated. During library construction, I have validated the TRIP framework using a variety of techniques (growth curves, qRT-PCR, and plasmid maintenance experiments). These studies indicate that stable expression of regulators is not broadly toxic, the selected promoter is inducible, and the construct vectors are stable both in vitro and in vivo. This project represents the first in vivo application of TRIP and will identify regulators and characterize key regulatory networks that drive Proteus mirabilis uropathogenesis.

抽象的 尿路感染（UTI）是常见感染，代表了医疗保健的重大负担 全球系统。仅在2006年，UTI是1100万医师访问的来源，花费了 美国超过35亿美元。这些感染的很大一部分是与导管相关的UTI （CAUTIS），占全球医院感染的40％。凯蒂斯的主要原因 是Proteus mirabilis，是肠杆菌命令的研究革兰氏阴性成员。这 有机体以其在感染患者的琼脂上蜂拥而至并形成尿石的能力最为著名。 先前的研究使用了uti的小鼠模型来识别有助于适应性和的因素 尿路中的毒力。尽管这些数据集强烈暗示转录调节器，但 了解该物种中的转录网络很多。为此，我已经开发了 采用转录调节剂引起的表型（TIP）筛选以识别的框架 有助于尿路病的特定调节剂。旅行利用包含压力的库 诱导表达构造每个编码单个调节器。这些菌株的池将是 接种到UTI小鼠模型中以评估比较适应性。测序和生物信息学 分析将用于评估行程菌株的相对适应性，并识别具有竞争性的菌株 膀胱环境中的边缘。确定这些关键调节器后，我的目标是定义下游 使用RNA测序并采用遗传方法，生化测定和 UTI的鼠模型，以确定健身优势背后的分子机制。使用 一种系统的生物信息学方法，我已经确定了232个假定的转录调节器 HI4320基因组。这些监管机构中只有3个定义了文献中发表的规范。到目前为止， 已经生成了35/232（15％）。在图书馆构造期间，我已经验证了 使用各种技术（生长曲线，QRT-PCR和质粒维持）的旅行框架 实验）。这些研究表明，调节剂的稳定表达不是广泛的毒性，而是 选定的启动子是可诱导的，并且构建体在体外和体内都是稳定的。这个项目 代表旅行的第一个体内应用，并将识别监管机构并表征密钥 驱动Proteus mirabilis泌尿病的调节网络。