Identification of bacterial small molecule inhibitors of Proteus mirabilis urease activity

奇异变形杆菌脲酶活性细菌小分子抑制剂的鉴定

• 批准号: 10609445
• 负责人: Chelsie Elizabeth Armbruster
• 金额: $ 19.94万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-13 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609445
• 关键词: Acids; Ammonia; Animals; Bacteremia; Bacteria; Bladder neck obstruction; Carbon Dioxide; Catheterization; Catheters; Cell Membrane Permeability; Cells; Clinical; Clinical Trials; Critical Illness; Cytoplasm; Data; Development; Enterobacter; Enterococcus; Enterococcus faecalis; Enzymes; Escherichia coli; Exhibits; FDA approved; Foundations; Future; Genes; Genetic Transcription; Goals; Gram-Negative Bacteria; Growth; Health; Holoenzymes; Human; In Vitro; Incidence; Incubated; Inductively Coupled Plasma Mass Spectrometry; Infection; Ions; Klebsiella aerogenes; Knowledge; Libraries; Life; Long-Term Care; Measures; Mediating; Mission; Modeling; Monitor; Morganella morganii; Multienzyme Complexes; Nickel; Operon; Pain; Pathway interactions; Patient Care; Patients; Post-Translational Protein Processing; Precipitation; Production; Protein Biosynthesis; Protein Import; Proteins; Proteus mirabilis; Providencia; Publications; Regimen; Regulation; Research; Risk; Risk Reduction; Secondary to; Sepsis; Severities; Structure-Activity Relationship; Therapeutic; Tissues; Toxic effect; Transcription Process; United States National Institutes of Health; Urea; Urease; Urinary Calculi; Urinary Catheterization; Urinary tract; Urine; Uropathogen; Western Blotting; candidate identification; catheter associated UTI; co-infection; enhancing factor; health care settings; healthcare-associated infections; high reward; high risk; inhibitor; innovation; insight; metabolomics; metalloenzyme; mortality; multi-drug resistant pathogen; mutant; novel; posttranscriptional; pre-clinical; prevent; renal damage; screening; side effect; signature molecule; small molecule; small molecule inhibitor; transcriptome sequencing; urinary; urolithiasis

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.

项目总结/摘要 留置导尿管的患者死亡率比未留置导尿管的患者高出近一倍 患者，部分原因是多药耐药微生物定植增加，导管相关尿路 感染（CITTI）和继发性菌血症。革兰氏阴性菌奇异变形杆菌是主要的 这可能是导致急性呼吸道感染和菌血症的原因，特别是长期导管插入。这种细菌长期以来 被认为是泌尿道的有问题的定植者，这是由于其有效的尿素酶，其水解 尿中的尿素转化为二氧化碳和氨。细菌尿素酶活性最终增加尿液pH值，诱导 多价离子的沉淀，并导致疼痛的导管结垢、堵塞和尿路结石 （尿石症）。在人类患者和动物感染模型中，奇异变形杆菌尿素酶活性升高膀胱 梗阻和肾损害，并大大促进菌血症的发展。然而，扰乱尿素酶 活性消除尿石症的发展，并显着降低菌血症的发病率， 尿素酶是治疗或预防奇异变形杆菌感染后遗症的一个有希望的靶点。此外，华为还经常 我们已经证明，奇异变形杆菌与其他尿路病原体的共感染调节了 尿石症和菌血症的风险，以尿素酶依赖的方式。具体而言，粪肠球菌和 斯氏普罗威登斯菌通过增强奇异普罗威登斯菌的尿素酶而增加尿石症和菌血症的发生率 活性，而摩根氏菌和产气肠杆菌通过抑制P. 紫茉莉脲酶活性在所有情况下，奇异变形杆菌尿素酶活性的调节是由尚未鉴定的 这些因子是分泌的，小于3 kDa，并且热稳定。因此，我们假设， 可以利用来自尿素酶调节物种的上清液来鉴定膜渗透性小的 在奇异变形杆菌中调节细胞质尿素酶活性的分子， 开发为治疗或预防奇异变形杆菌感染后遗症的非抗生素方法。在目标1中， 我们将确定通过无细胞上清液调节尿素酶的作用机制，重点是i）直接 与奇异变形杆菌尿素酶的相互作用和ii）对尿素酶结构亚基产生的间接影响 以及催化活性脲酶全酶与脱辅基酶的比例。在目标2中，我们将在全球范围内 代谢组学研究，以鉴定来自 抑制奇异变形杆菌脲酶活性。通过这项研究揭示的候选小分子将被 购买，合成，分离或富集，以验证他们减少奇异变形杆菌的机制 脲酶活性所提出的方法代表了在寻求以下方面的概念和技术创新： 脲酶抑制剂，并有可能改变模式的奇异变形杆菌脲酶调节。知识 获得的将支持未来的R 01，旨在评估脲酶抑制小分子的治疗潜力， 分子和开发临床前方案，以降低动物感染模型中的PTI后遗症的风险。