Division of Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
Antimicrob Agents Chemother. 2019 Aug 23;63(9). doi: 10.1128/AAC.00730-19. Print 2019 Sep.
Bacteria have acquired multiple mechanisms to evade the lethal effects of current therapeutics, hindering treatment of bacterial infections, such as those caused by the pathogen , which is responsible for nosocomial and cystic fibrosis lung infections. One resistance mechanism involves membrane-embedded multidrug efflux pumps that can effectively extrude an array of substrates, including common antibiotics, dyes, and biocides. Among these is a small multidrug resistance (SMR) efflux protein, consisting of four transmembrane (TM) helices, that functions as an antiparallel dimer. TM helices 1 to 3 (TM1 to TM3) comprise the substrate binding pocket, while TM4 contains a GG7 heptad sequence motif that mediates the SMR TM4-TM4 dimerization. In the present work, we synthesized a series of peptides containing the residues centered on the TM4-TM4 binding interface found in the SMR (PAsmr), typified by Ac-Ala-(Sar)-LLGIALV-KKK-NH (helix-helix interaction residues are underlined). Here, the acetylated N-terminal sarcosine (-methyl-Gly) tag [Ac-Ala-(Sar)] promotes membrane penetration, while the C-terminal Lys tag promotes selectivity for the negatively charged bacterial membranes. This peptide was observed to competitively disrupt PAsmr-mediated efflux, as measured by efflux inhibition of the fluorescent dye ethidium bromide, while having no effect on cell membrane integrity. Alternatively, a corresponding peptide in which the TM4 binding motif is scrambled was inactive in this assay. In addition, when cells expressing PAsmr were combined with sublethal concentrations of several biocides, growth was significantly inhibited when peptide was added, notably, by up to 95% with the disinfectant benzylalkonium chloride. These results demonstrate promise for an efflux pump inhibitor to address the increasing threat of antibiotic-resistant bacteria.
细菌已经获得了多种逃避现有治疗方法致命作用的机制，这阻碍了细菌感染的治疗，例如由病原体引起的感染，该病原体可导致医院获得性和囊性纤维化肺部感染。一种耐药机制涉及膜嵌入式多药外排泵，可有效地排出一系列底物，包括常见的抗生素、染料和杀生剂。其中包括一个小的多药耐药（SMR）外排蛋白，由四个跨膜（TM）螺旋组成，作为反平行二聚体发挥作用。TM 螺旋 1 到 3（TM1 到 TM3）组成了底物结合口袋，而 TM4 包含一个 GG7 七肽序列基序，介导 SMR TM4-TM4 二聚化。在本工作中，我们合成了一系列含有 SMR 中 TM4-TM4 结合界面中心残基的肽（PAsmr），其典型代表是 Ac-Ala-（Sar）-LLGIALV-KKK-NH（螺旋-螺旋相互作用残基下划线）。这里，乙酰化的 N 端肌氨酸（-甲基-Gly）标签[Ac-Ala-（Sar）]促进膜穿透，而 C 端赖氨酸标签促进对带负电荷的细菌膜的选择性。如通过荧光染料溴化乙锭的外排抑制来测量，该肽观察到可竞争性地破坏 PAsmr 介导的外排，而对细胞膜完整性没有影响。或者，该 TM4 结合基序被打乱的对应肽在该测定中是无活性的。此外，当表达 PAsmr 的细胞与亚致死浓度的几种杀生剂结合时，当添加肽时，生长明显受到抑制，值得注意的是，用消毒剂苯扎氯铵可高达 95%。这些结果表明，外排泵抑制剂有望解决抗生素耐药菌日益增加的威胁。
