Systematic analysis of Proteus mirabilis transcriptional regulators that drive uropathogenesis

摘要 尿路感染（UTI）是常见的感染，对医疗保健造成重大负担 全球系统。仅在2006年，UTI就有1100万人次就诊， 美国超过35亿美元。这些感染中很大一部分是导管相关性尿路感染 （CIMTI），占全球医院获得性感染的40%。一个主要的原因， 奇异变形杆菌是肠球菌目中一种研究不足的革兰氏阴性菌。这 这种微生物以其在琼脂上聚集并在受感染的患者中形成泌尿系统结石的能力而闻名。 以前的研究使用了UTI的小鼠模型来确定有助于健康和 泌尿道的毒性。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还有很多要学。为此，我制定了 框架采用转录调节诱导表型（TRIP）筛选，以确定 有助于泌尿发病的特定调节剂。TRIP利用了一个菌株库， 可诱导表达构建体，每个构建体编码单个调节子。这些菌株中的20%将是 接种到UTI的小鼠模型中以评估比较适合性。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。在确定这些关键监管机构后，我的目标是定义下游 使用RNA测序的调控网络，并采用遗传方法，生物化学测定， UTI的小鼠模型，以确定适应性优势背后的分子机制。使用 一个系统的生物信息学方法，我已经确定了232个假定的转录调节因子， HI 4320基因组。这些调节子中只有3个在文献中公开定义了调节子。到目前为止， 35/232（15%）的构建体已经生成。在图书馆建设过程中，我验证了 使用各种技术（生长曲线、qRT-PCR和质粒维持）的TRIP框架 实验）。这些研究表明，稳定表达的调节剂不是广泛毒性的， 选择的启动子是可诱导的，并且构建载体在体外和体内都是稳定的。这个项目 代表了TRIP的首次体内应用，并将确定监管机构和表征关键 调控网络驱动奇异变形杆菌尿路发病机制。

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.