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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

基本信息

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
项目类别：
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

项目摘要

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