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: 10386534
• 负责人: Madison Jane Fitzgerald
• 金额: $ 4.04万
• 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/10386534
• 关键词: Accounting; Address; 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; Foundations; Future; Genes; Genetic Transcription; Genome; Goals; Growth; 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; 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; crystallinity; 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，给医疗保健带来重大负担 世界各地的系统。仅 2006 年，就有 1100 万人次因尿路感染就诊，并花费了 美国超过35亿美元。这些感染的很大一部分是导管相关性尿路感染 (CAUTI)，占全球医院获得性感染的 40%。 CAUTI 的一个主要原因 奇异变形杆菌是一种尚未被研究的肠杆菌目革兰氏阴性成员。这 该生物体最著名的是它能够聚集在琼脂上并在感染患者体内形成尿结石。 先前的研究已经使用尿路感染小鼠模型来识别有助于健康和健康的因素。 泌尿道的毒力。虽然这些数据集强烈暗示转录调节因子，但 关于这个物种的转录网络还有很多值得了解的地方。为此，我开发了 框架采用转录调节子诱导表型 (TRIP) 筛选来识别 有助于尿路病理发生的特定调节因子。 TRIP 利用菌株库，其中包含 诱导型表达构建体各自编码一个调节因子。这些菌株的池将 接种到尿路感染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 TRIP 菌株的相对适应性并鉴定具有竞争性的菌株 膀胱环境的边缘。确定这些关键监管机构后，我的目标是定义下游 使用 RNA 测序并采用遗传方法、生化检测和 UTI 小鼠模型以确定健康优势背后的分子机制。使用 通过系统的生物信息学方法，我在 HI4320 基因组。这些调节器中只有 3 个在文献中发表了调节子的定义。到目前为止， 35/232 (15%) 的构建体已生成。在图书馆建设过程中，我验证了 TRIP 框架使用多种技术（生长曲线、qRT-PCR 和质粒维护） 实验）。这些研究表明，调节因子的稳定表达并不具有广泛的毒性， 所选启动子是可诱导的，并且构建的载体在体外和体内均稳定。这个项目 代表 TRIP 的首次体内应用，并将识别调节剂并表征关键 驱动奇异变形杆菌尿路病理发生的调控网络。