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

摘要 抗生素耐药性是一个日益增长的全球健康问题，部分原因是由于外排泵的作用， 病原体一类外排泵，小多药耐药转运蛋白（SMR），清除有毒物质， 化合物从细胞与质子耦合运输。SMR在历史上被描述为反向转运体， 但最近的证据表明，最好的研究SMR，EmrE，可以执行反向转运，同向转运， 和/或基于“自由交换”模型的单端口。该模型表明，SMRs可能诱导对 一些化合物，而不是电阻，无论是通过直接流入/共输或通过质子的耗尽， 通过不受控制的质子单端口提供动力。在任何一种情况下，这都是一个强大的策略，因为它需要一个 SMR仅仅是存在的，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）是其他多药耐药外排的主要能量来源， 由于PMF是一种外排泵，PMF的减少意味着针对其他外排泵，而不仅仅是SMR。在此，我提议 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可能诱导易感性，而不是耐药性，一些， 化合物.目的1：利用表型微阵列技术发现PaSMR的新底物，并进行验证 增长曲线。将比较WT PaSMR和转运死亡突变体，以确定这些底物 触发电阻或敏感性。在目标2中，基于固体支撑膜的电生理学实验 将揭示基于不同基质/质子梯度的传输电荷差异的传输模式。 这被假设为电阻基板的反向端口，但可能是同向端口或单端口的敏感性 印刷受体.最后，在目标3中，将确定PaSMR的溶液NMR共振分配，允许 跟踪特定残基和与不同底物的结合相互作用。这将确定具体的 负责易感性结果的相互作用。整体而言，这项建议将改变我们的运输模式， 揭示PaSMR如何以基质依赖性方式改变运输模式，并研究诱导 敏感性和使用质子动力作为多重耐药感染的治疗途径。 这个培训计划将发展我的微生物学知识和技术， 健康问题、生物物理技术和实验设计，以及大型 数据集。研究将在威斯康星大学麦迪逊分校进行，这是一项领先的生物化学研究。 中心，在凯瑟琳亨茨勒-怀德曼博士的监督下，在该领域的著名研究员， 运输以及麦迪逊国家磁共振设施的联合主任。培训将采取 放置在生物化学综合计划，提供高质量的生物化学教育， 在负责任的研究行为的培训，和专业发展的机会，准备我是一个 传染病研究的未来领导者

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.