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

• 批准号: 10461633
• 负责人: Andrea Killian Wegrzynowicz
• 金额: $ 3.43万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 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/10461633
• 关键词: Antibiotic Resistance; Antibiotics; Behavior; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Assay; Biological Models; Cell Respiration; Cells; Charge; Chemicals; Clinical; Coupled; Coupling; Data; Development; Dose; 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; Microbiology; 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; Training and Education; Transport Process; Uniport; Universities; Vertebral column; Wisconsin; antiport; antiporter; base; 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

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.

摘要 抗生素耐药性是一个日益严重的全球健康问题，部分原因是外排泵在体内的作用 病原体。一类外排泵，小型多药耐药转运体(SMR)，清除有毒物质 化合物从细胞中以质子耦合的方式传输。SMR历来被描述为反转运蛋白， 但最近的证据表明，研究最好的SMR，EmRE，可以执行反移植，符号移植， 和/或基于“自由交换”模式的Uniport。这一模型表明SMR可能诱导易感性 一些化合物而不是阻力，要么是通过直接流入/转移，要么是通过质子的耗尽- 动力来自不受控制的质子港。无论是哪种情况，这都是一种强大的策略，因为它需要 SMR仅仅存在，而不是给定细菌种群的主要耐药机制。 此外，由于质子动力(PMF)是其他多药耐药外排的主要能源 泵，PMF的关闭意味着瞄准其他外排泵，而不仅仅是SMR。在此，我提出一个 假单胞菌EmRE同源物PaSMR转运机制的研究 铜绿假单胞菌，假设PaSMR可能会诱导对某些 化合物。在目标1中，将通过表型微阵列发现并验证PaSMR的新底物 通过生长曲线。WT PaSMR和一个运输死亡突变体将被比较以确定这些底物是否 触发电阻或敏感度。在目标2中，基于固体支撑膜的电生理实验 将根据不同衬底/质子梯度的传输电荷的不同来揭示传输模式。 这被假设为与抗性底物相反，但可能是敏感性的符号或唯一端口 底物。最后，在目标3中，将确定PaSMR的溶液核磁共振共振分配，从而允许 跟踪特定残基和与不同底物的结合作用。这将标识特定的 相互作用是导致易感性结果的原因。总体而言，这项建议将通过以下方式改变我们的交通模式 揭示PaSMR如何以底物依赖的方式改变运输方式，并研究诱导 敏感性和使用质子动力衰竭作为多药耐药感染的治疗途径。 这项培训计划将发展我的微生物知识和技术，了解公众 关注健康、生物物理技术和实验设计，以及管理和解释 数据集。这项研究将在威斯康星大学麦迪逊分校进行，这是一项领先的生化研究 中心，由Katherine Henzler-Wildman博士监督，Katherine Henzler-Wildman博士是该领域的著名研究员 他也是位于麦迪逊的国家磁共振设施的联合主任。培训将需要 在提供高质量生物化学教育的生物化学综合课程中占有一席之地， 在负责任的研究和职业发展机会方面的培训，为我成为一名 未来传染病研究的领导者。