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.

摘要 尿路感染(UTI)是一种常见的感染，给医疗保健带来了巨大的负担 世界各地的系统。仅在2006年，尿路感染就是1100万医生就诊的来源，并使 美国超过35亿美元。这些感染中很大一部分是与导管相关的尿路感染 (CAUTIs)，占全球医院获得性感染的40%。CAUTIs的一个主要原因 是奇异变形杆菌，是肠杆菌目中一种未被充分研究的革兰氏阴性菌。这 细菌最著名的是它在琼脂上聚集并在感染患者身上形成尿结石的能力。 以前的研究已经使用尿路感染的小鼠模型来确定有助于健康和 尿路中的毒力。虽然这些数据集强烈暗示了转录调控因子，但 关于这个物种的转录网络有很多需要了解的。为此，我开发了 利用转录调节因子诱导表型(TRIP)筛查进行鉴定的框架 导致尿路致病的特定调节因子。TRIP利用了一个包含以下内容的菌株库 可诱导表达构建体，每个表达构建体编码一个单一的调节子。这些菌株的池将是 接种于UTI小鼠模型，评估其相对适合性。测序和生物信息学 分析将用于评估TRIP菌株的相对适合度，并鉴定具有竞争性的菌株 在膀胱环境中的边缘。在确定了这些关键监管机构之后，我的目标是定义下游 使用RNA测序的调控网络，并使用遗传方法、生化分析和 UTI的小鼠模型，以确定健康优势背后的分子机制。vbl.使用 通过系统的生物信息学方法，我已经确定了232个假定的转录调控因子 HI4320基因组。在这些监管机构中，只有3家定义了文献中公布的监管规定。到目前为止， 已经生成了35/232(15%)的构建体。在图书馆建设过程中，我已经验证了 使用多种技术的TRIP框架(生长曲线、qRT-PCR和质粒维护 实验)。这些研究表明，稳定表达调节剂没有广泛的毒性， 所选择的启动子是可诱导的，构建的载体在体内外都是稳定的。这个项目 代表了TRIP的第一个活体应用，并将确定调节器和表征关键 驱动奇异变形杆菌泌尿致病的调控网络。