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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/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，部分原因是多重耐药微生物、导管相关尿路定植增加感染（CAUTI）和继发性菌血症。革兰氏阴性菌奇异变形杆菌是主要的细菌引起 CAUTI 和菌血症，尤其是长期导尿时。这种细菌长期以来由于其强大的脲酶可水解尿路，因此被认为是泌尿道的有问题的定植者。尿液中的尿素转化为二氧化碳和氨。细菌脲酶活性最终增加尿液 pH 值，诱导多价离子沉淀，导致痛苦的导管结垢、堵塞和尿路结石（尿石症）。在人类患者和动物感染模型中，奇异果脲酶活性引发膀胱阻塞和肾损伤，并极大地促进菌血症的发展。然而，破坏脲酶活性消除尿石症的发展并显着降低菌血症的发生率，使得脲酶是治疗或预防奇异果 P. mirabilis CAUTI 后遗症的一个有前景的靶点。 CAUTI 也经常发生多种微生物，我们已经证明奇异变形杆菌与其他尿路病原体的共同感染会调节以尿素酶依赖性方式产生尿石症和菌血症的风险。具体而言，粪肠球菌和斯氏普罗威登斯菌通过增强奇异果脲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疟原虫来降低感染的严重程度。奇异果脲酶活性。在所有情况下，奇异果脲酶活性的调节是由尚未鉴定的分泌的因子，小于 3 kDa，且热稳定。因此我们假设无细胞脲酶调节物种的上清液可用于鉴定膜渗透性小分子调节奇异果细胞质脲酶活性的分子，具有强大的潜力被开发成一种非抗生素方法，用于治疗或预防奇异变形杆菌 CAUTI 后遗症。在目标 1 中，我们将确定无细胞上清液调节脲酶的作用机制，重点是 i) 直接与奇异果脲酶的相互作用以及 ii) 对脲酶结构亚基产生的间接影响以及具有催化活性的脲酶全酶与脱辅基酶的比率。在目标 2 中，我们将开展全球代谢组学研究，鉴定来自以下物种的无细胞上清液特有的小分子特征：抑制奇异果脲酶活性。通过这项研究揭示的候选小分子将被购买、合成、分离或富集以验证它们减少奇异果的机制脲酶活性。所提出的方法代表了寻找方法的概念和技术创新。脲酶抑制剂，并有可能改变奇异果脲酶调节的范式。知识点获得的成果将支持未来的 R01，旨在评估脲酶抑制小分子的治疗潜力分子并开发临床前治疗方案以降低动物感染模型中 CAUTI 后遗症的风险。