Inducing susceptibility with a small multidrug resistance transporter from P. aeruginosa

用来自铜绿假单胞菌的小型多药耐药转运蛋白诱导敏感性

基本信息

批准号：
10619555
负责人：
Andrea Killian Wegrzynowicz
金额：
$ 3.53万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
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：
2022-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10619555
关键词：
Antibiotic Resistance Antibiotics Behavior Binding Binding Sites Biochemical Biochemistry Biological Biological Assay Biological Models Cell Respiration Cells Charge Chemicals Classification Clinical Coupled Coupling Data Development Dose Education Electrophysiology (science)Energy-Generating Resources Escherichia coli Experimental Designs Foundations Future Goals Growth Homologous Gene Infection Infectious Diseases Research Investigation Ion Cotransport Knowledge Lead Liposomes Local Anti-Infective Agents Magnetic Resonance Membrane Minimum Inhibitory Concentration measurement Modeling Molecular Monitor Multi-Drug Resistance Mutation Natural Products Organism Outcome Pharmaceutical Preparations Phenotype Plants Poison Population Predisposition Proteins Proton-Motive Force Protons Pseudomonas aeruginosa Public Health Research Research Personnel Resistance Respiration Site Solid Substrate Interaction Supervision Techniques Testing Therapeutic Training Transport Process Uniport Universities Vertebral column Wisconsin antiport antiporter biophysical techniques clinically relevant cystic fibrosis patients efflux pump experimental study global health human pathogen in vivo innovation large datasets mutant novel novel therapeutics pathogen priority pathogen programs resistance mechanism responsible research conduct small molecule uptake

项目摘要

ABSTRACT Antibiotic resistance is a growing global health concern, due in part to the action of efflux pumps in pathogens. One class of efflux pumps, the Small Multidrug Resistance transporters (SMRs), remove toxic compounds from the cell with proton-coupled transport. SMRs have historically been described as antiporters, but recent evidence demonstrates that the best-studied of the SMRs, EmrE, can perform antiport, symport, and/or uniport based on a “free-exchange” model. This model suggests that SMRs may induce susceptibility to some compounds rather than resistance, either through direct influx/symport or by rundown of the proton- motive force through uncontrolled proton uniport. In either case, this is a powerful strategy as it requires an SMR to be merely present, rather than be the primary resistance mechanism of the given bacterial population. Additionally, as the proton-motive force (PMF) is the main energy source of other multidrug-resistance efflux pumps, rundown of the PMF means targeting other efflux pumps, not just SMRs. Herein I propose an investigation of the transport mechanisms of PaSMR, an EmrE homolog from the pathogen Pseudomonas aeruginosa, hypothesizing that PaSMR may induce susceptibility, rather than resistance, to some compounds. In Aim 1, novel substrates of PaSMR will be discovered by phenotypic microarray and validated by growth curves. WT PaSMR and a transport-dead mutant will be compared to determine if these substrates trigger resistance or susceptibility. In Aim 2, solid-supported membrane-based electrophysiology experiments will reveal transport mode based on differences in transported charge with various substrate/proton gradients. This is hypothesized to be antiport for resistance substrates, but may be symport or uniport for susceptibility substrates. Finally, in Aim 3, solution NMR resonance assignments for PaSMR will be determined, allowing the tracking of specific residues and binding interactions with different substrates. This will identify specific interactions responsible for susceptibility outcomes. Overall, this proposal will shift our paradigm of transport by uncovering how PaSMR changes transport mode in a substrate-dependent manner, and investigate inducing susceptibility and using proton-motive force rundown as a therapeutic avenue for multidrug-resistant infections. This training plan will develop my microbiological knowledge and techniques, understanding of public health concerns, biophysical techniques and experimental design, and management and interpretation of large data sets. Research will be conducted at the University of Wisconsin-Madison, a leading biochemical research center, under the supervision of Dr. Katherine Henzler-Wildman, a renowned researcher in the field of transport as well as a co-director of the National Magnetic Resonance Facility at Madison. Training will take place within the Integrated Program in Biochemistry, which provides high-quality biochemical education, training in responsible conduct of research, and professional development opportunities to prepare me to be a future leader in infectious disease research.

抽象的抗生素耐药性是一个日益严重的全球健康问题，部分原因是外排泵的作用一类外排泵，即小型多药耐药转运蛋白（SMR），可以清除有毒物质。来自具有质子耦合转运的细胞的化合物历来被描述为反向转运蛋白，但最近的证据表明，SMR 中研究最充分的 EmrE 可以执行反移植、同向移植、和/或基于“自由交换”模型的单端口该模型表明 SMR 可能会诱发对病毒的易感性。一些化合物而不是阻力，无论是通过直接流入/同向运输还是通过质子的耗尽无论哪种情况，这都是一个强大的策略，因为它需要一个强大的策略。 SMR 只是存在，而不是给定细菌群体的主要耐药机制。此外，由于质子动力（PMF）是其他多重耐药外排的主要能量来源泵，PMF 的淘汰意味着针对其他外排泵，而不仅仅是 SMR。 PaSMR（来自病原体假单胞菌的 EmrE 同源物）转运机制的研究铜绿假单胞菌，假设 PaSMR 可能会诱导对某些细菌的敏感性，而不是耐药性在目标 1 中，将通过表型微阵列发现 PaSMR 的新底物并进行验证。通过生长曲线比较 WT PaSMR 和转运死亡突变体，以确定这些底物是否有效。在目标 2 中，基于固体支持的膜的电生理学实验。将揭示基于不同底物/质子梯度的传输电荷差异的传输模式。这被认为是抗性底物的反端口，但对于敏感性可能是同向端口或单端口最后，在目标 3 中，将确定 PaSMR 的溶液 NMR 共振分配，从而允许跟踪特定残基以及与不同底物的结合相互作用，这将识别特定的残基。总体而言，该提案将通过以下方式改变我们的运输范式。揭示 PaSMR 如何以底物依赖性方式改变传输模式，并研究诱导敏感性并使用质子动力下降作为多重耐药感染的治疗途径。该培训计划将发展我的微生物知识和技术，了解公众健康问题、生物物理技术和实验设计以及大型研究的管理和解释研究将在威斯康星大学麦迪逊分校进行，这是一家领先的生化研究机构。中心，在该领域的著名研究员 Katherine Henzler-Wildman 博士的指导下运输以及麦迪逊国家磁共振设施的联合主任将负责。在生物化学综合项目中占有一席之地，该项目提供高质量的生物化学教育，负责任的研究行为培训和专业发展机会，使我做好成为一名传染病研究的未来领导者。