Harnessing polymicrobial interactions in the catheterized urinary tract to identify novel inhibitors of Proteus mirabilis urease activity

项目摘要 导管相关性尿路感染(CAUTI)是继发医院血流的主要原因 感染。产生尿素酶的微生物，如奇异变形杆菌，是引起CAUTI和 伴随着许多并发症。尿素酶将尿素分解成氨，从而提高尿液的pH值和 导致离子沉淀到晶体中，最终导致导尿管结垢和堵塞以及尿路 结石的形成和增加菌血症、败血症和死亡的风险。唯一被批准的尿素酶抑制剂 美国食品和药物管理局(FDA)--乙酰异羟肟酸(AHA)--显示出预防尿路感染的效果 结石形成，但有严重的副作用，限制了其临床应用。因此，替代战略是 用于靶向细菌尿素酶活性，以防止导管结垢和结石形成。在病人中 对于长期留置导尿管，CAUTI通常是多菌的。正如我们以前的出版物所展示的那样 和初步数据，我们发现多菌CAUTI的共同成分分泌分子 调节奇异疫霉的尿素酶活性和感染严重程度。因此，我们执行了无目标的全局 尿素酶调控菌无细胞培养上清液的代谢组学分析 候选尿素酶抑制代谢物。我们验证了四个化合物的活性：组胺(CAS 51-45- 6)、亮甘氨酸(CAS 686-50-0)、苯丙酮酸(CAS 156-06-9)和咪唑乳酸(CAS 14403-45-3)。 我们的初步数据表明，亮氨酸甘氨酸和咪唑乳酸至少在一定程度上抑制了尿素酶的活性， 通过直接作用于尿素酶活性部位。相反，组胺和苯丙酮酸似乎间接地 通过一种未知的机制抑制尿素酶的活性。此NRSA F30计划的目标是 确定尿素酶调节的作用机制，并进行临床前评估 这些抑制化合物的治疗潜力。目标1将确定间接尿素酶的作用机制 并探讨不同作用机制的化合物对组胺和苯丙酮酸的调节作用 当与一组尿素酶联合给药时，作用对尿素酶活性有协同作用。 产生病原体的。目标2将评估抑制化合物的翻译潜力，以防止 Foley导尿管结垢、结晶生物膜形成和堵塞。这篇文章中概述的实验 该提案将提供一个框架，利用多菌相互作用来进行药物发现和 开发新的治疗方法以预防CAUTI相关的发病率和死亡率。这项工作将需要 在雅各布斯医学院和生物医学科学学院切尔西·阿姆布鲁斯特博士的实验室里， 世卫组织是CAUTI和微生物发病机制研究方面的专家。培训计划是为我的 作为传染病领域的内科科学家的发展，包括由成功的 传染病和内科的内科科学家和临床导师。

Project Summary Catheter-associated urinary tract infection (CAUTI) is the leading cause of secondary nosocomial bloodstream infections. Urease-producing organisms such as Proteus mirabilis are common causes of CAUTI and associated with numerous complications. Urease hydrolyzes urea to ammonia, which raises the urine pH and causes precipitation of ions into crystals, ultimately leading to catheter encrustation and blockage and urinary stone formation and increasing the risk of bacteremia, sepsis and death. The only urease inhibitor approved by the Food and Drug Administration (FDA), acetohydroxamic acid (AHA), shows efficacy in preventing urinary stone formation but has severe side effects that limit its clinical use. Therefore, alternative strategies are needed for targeting bacterial urease activity to prevent catheter encrustation and stone formation. In patients with long-term indwelling catheters, CAUTI is often polymicrobial. As demonstrated in our prior publications and preliminary data, we found that common constituents of polymicrobial CAUTI secrete molecules that modulate P. mirabilis urease activity and infection severity. We therefore performed untargeted global metabolomics analysis on cell-free supernatants of urease-modulating bacterial strains and identified 36 candidate urease dampening metabolites. We validated the activity of four compounds: histamine (CAS 51-45- 6), leucylglycine (CAS 686-50-0), phenylpyruvate (CAS 156-06-9) and imidazole lactate (CAS 14403-45-3). Our preliminary data suggests that leucylglycine and imidazole lactate dampen urease activity, at least in part, by acting directly on the urease active site. In contrast, histamine and phenylpyruvate appear to indirectly inhibit urease activity through an uncharacterized mechanism. The goal of this NRSA F30 proposal is to determine the mechanism of action of urease modulation and conduct pre-clinical assessment of the therapeutic potential of these dampening compounds. Aim 1 will determine the mechanism of indirect urease modulation for histamine and phenylpyruvate and explore whether compounds of different mechanisms of action have synergistic effects on urease activity when administered in combination against a panel of urease- producing pathogens. Aim 2 will assess the translational potential of dampening compounds for preventing Foley catheter encrustation, crystalline biofilm formation, and blockage. The experiments outlined in this proposal will provide a framework for harnessing polymicrobial interactions for drug discovery and the development of novel therapeutics to prevent CAUTI associated morbidity and mortality. This work will take place at the Jacobs School of Medicine and Biomedical Sciences in the laboratory of Dr. Chelsie Armbruster, who is an expert in CAUTI and microbial pathogenesis research. The training plan is tailored for my development as a physician-scientist in the field of infectious disease, and includes mentoring by successful physician-scientists and clinical preceptorships in infectious disease and internal medicine.