Identification of bacterial small molecule inhibitors of Proteus mirabilis urease activity

项目摘要/摘要 留置导尿管患者的死亡率几乎是未留置导尿管患者的两倍 患者，部分原因是耐多药细菌的定植增加，导尿管相关的尿路 感染(CAUTI)和继发性菌血症。革兰氏阴性菌奇异变形杆菌是一种优势菌 CAUTI和菌血症的原因，特别是长期导尿。这种细菌长期以来一直是 被认为是尿路的一种有问题的定殖者，因为它有强大的尿素酶，可以分解尿路中的 尿液中的尿素转化为二氧化碳和氨。细菌尿素酶活性最终会增加尿液的pH值，诱导 多价离子的沉淀，并导致痛苦的导尿管结垢、堵塞和尿路结石 (尿石症)。在人类患者和动物感染模型中，奇异肺炎杆菌尿素酶活性引起膀胱 梗阻和肾脏损害，极大地促进了菌血症的发展。然而，扰乱尿素酶 活动消除了尿石症的发展，并显著降低了菌血症的发生率，使 尿素酶是治疗或预防奇异肺炎后遗症的有希望的靶点。CAUTI也经常 我们已经证明了奇异肺炎杆菌与其他泌尿系统病原体的混合感染调节了 尿素酶依赖的尿石症和菌血症的风险。具体地说，粪肠球菌和 普氏菌通过增强奇异杆菌尿素酶增加尿石症和菌血症的发生率 摩根摩根氏菌和产气肠杆菌通过抑制P。 芒果尿素酶活性。在所有情况下，调节奇异P.mirabilis尿素酶活性是由迄今尚未确定的 分泌的、小于3 kDa的、热稳定的因子。因此，我们假设无细胞 来自尿素酶调节物种的上清液可以用来识别膜透性小分子 调节奇异青霉胞质尿素酶活性的分子，具有很强的潜力 发展成为治疗或预防奇异肺炎后遗症的非抗生素方法。在目标1中， 我们将确定无细胞培养上清液调节尿素酶的作用机制，重点是i)直接 与奇异假单胞菌尿素酶的相互作用及II)对尿素酶结构亚基产生的间接影响 以及具有催化活性的尿素酶全酶与酶的比值。在目标2中，我们将进行一次全球 代谢组学研究，以确定来自物种的无细胞培养上清液特有的小分子特征 抑制奇异拟青霉的尿素酶活性。通过这项研究揭示的候选小分子将是 购买、合成、分离或浓缩，以验证它们减少奇异疫霉的机制 尿素酶活性。拟议的方法是一项概念和技术创新，旨在寻找 尿素酶抑制剂，并有可能改变奇异假单胞菌尿素酶调节的范式。《知识》 Gain将支持未来的R01，旨在评估尿素酶抑制Small的治疗潜力 分子，并开发临床前方案，以降低动物感染模型中CAUTI后遗症的风险。

Project Summary/Abstract Patients with indwelling urinary catheters have nearly double the mortality rate compared to non-catheterized patients, in part due to increased colonization by multidrug-resistant organisms, catheter-associated urinary tract infections (CAUTI), and secondary bacteremia. The Gram-negative bacterium Proteus mirabilis is a predominant cause of both CAUTI and bacteremia, particularly with long-term catheterization. This bacterium has long been recognized as a problematic colonizer of the urinary tract due to its potent urease enzyme, which hydrolyzes the urea in urine to carbon dioxide and ammonia. Bacterial urease activity ultimately increases urine pH, induces precipitation of polyvalent ions, and causes painful catheter encrustation, blockage, and urinary stones (urolithiasis). In human patients and animal infection models, P. mirabilis urease activity elicits bladder obstruction and renal damage and greatly facilitates the development of bacteremia. However, disrupting urease activity abrogates development of urolithiasis and dramatically reduces the incidence of bacteremia, making urease a promising target for treating or preventing P. mirabilis CAUTI sequelae. CAUTI is also frequently polymicrobial, and we have demonstrated that co-infection of P. mirabilis with other uropathogens modulates the risk of urolithiasis and bacteremia in a urease-dependent manner. Specifically, Enterococcus faecalis and Providencia stuartii increase the incidence of urolithiasis and bacteremia by enhancing P. mirabilis urease activity, while Morganella morganii and Enterobacter aerogenes decrease infection severity by dampening P. mirabilis urease activity. In all cases, modulation of P. mirabilis urease activity is mediated by as-yet unidentified factors that are secreted, smaller than 3 kDa, and heat-stable. We therefore hypothesize that cell-free supernatants from urease-modulatory species can be exploited to identify membrane-permeable small molecules that regulate activity of the cytoplasmic urease enzyme in P. mirabilis and that have strong potential to be developed into a non-antibiotic approach for treating or preventing P. mirabilis CAUTI sequelae. In Aim 1, we will determine the mechanism of action of urease modulation by cell-free supernatants focusing on i) direct interaction with the P. mirabilis urease enzyme and ii) indirect effects on production of urease structural subunits and the ratio of catalytically-active urease holoenzyme to apoenzyme. In Aim 2, we will conduct a global metabolomics study to identify small molecule signatures unique to cell-free supernatants from species that dampen P. mirabilis urease activity. Candidate small molecules revealed through this study will then be purchased, synthesized, isolated, or enriched for to verity the mechanism by which they decrease P. mirabilis urease activity. The proposed approach represents both a conceptual and technical innovation in the search for urease inhibitors, and has the potential to change the paradigm of P. mirabilis urease regulation. The knowledge gained will support a future R01 aimed at assessing the therapeutic potential of urease-dampening small molecules and developing a pre-clinical regimen to reduce risk of CAUTI sequelae in animal infection models.