Data_Sheet_1_Exposure to Sub-inhibitory Concentrations of the Chemosensitizer 1-(1-Naphthylmethyl)-Piperazine Creates Membrane Destabilization in Multi-Drug Resistant Klebsiella pneumoniae.PDF

Antimicrobial efflux is one of the important mechanisms causing multi-drug resistance (MDR) in bacteria. Chemosensitizers like 1-(1-naphthylmethyl)-piperazine (NMP) can inhibit an efflux pump and therefore can overcome MDR. However, secondary effects of NMP other than efflux pump inhibition are rarely investigated. Here, using phenotypic assays, phenotypic microarray and transcriptomic assays we show that NMP creates membrane destabilization in MDR Klebsiella pneumoniae MGH 78578 strain. The NMP mediated membrane destabilization activity was measured using β-lactamase activity, membrane potential alteration studies, and transmission electron microscopy assays. Results from both β-lactamase and membrane potential alteration studies shows that both outer and inner membranes are destabilized in NMP exposed K. pneumoniae MGH 78578 cells. Phenotypic Microarray and RNA-seq were further used to elucidate the metabolic and transcriptional signals underpinning membrane destabilization. Membrane destabilization happens as early as 15 min post-NMP treatment. Our RNA-seq data shows that many genes involved in envelope stress response were differentially regulated in the NMP treated cells. Up-regulation of genes encoding the envelope stress response and repair systems show the distortion in membrane homeostasis during survival in an environment containing sub-inhibitory concentration of NMP. In addition, the lsr operon encoding the production of autoinducer-2 responsible for biofilm production was down-regulated resulting in reduced biofilm formation in NMP treated cells, a phenotype confirmed by crystal violet-based assays. We postulate that the early membrane disruption leads to destabilization of inner membrane potential, impairing ATP production and consequently resulting in efflux pump inhibition.

抗菌药物外排是导致细菌多重耐药（MDR）的重要机制之一。诸如1-(1-萘甲基)-哌嗪（NMP）这类化学增敏剂可通过抑制外排泵功能进而克服细菌的多重耐药性。然而，目前针对NMP除外排泵抑制之外的次级效应的相关研究仍较为匮乏。本研究借助表型分析、表型微阵列及转录组分析技术，证实NMP可在多重耐药肺炎克雷伯菌MGH 78578菌株中诱导膜去稳定化现象。研究通过β-内酰胺酶活性检测、膜电位变化分析及透射电子显微镜实验，对NMP介导的膜去稳定化活性进行了测定。结果显示，经NMP处理的肺炎克雷伯菌MGH 78578细胞的外膜与内膜均发生去稳定化，该结论得到了β-内酰胺酶活性与膜电位变化两组实验结果的共同验证。此外，本研究结合表型微阵列与RNA测序（RNA-seq）技术，进一步解析了支撑膜去稳定化过程的代谢与转录调控信号。膜去稳定化现象可在NMP处理后15分钟内即被观测到。RNA-seq数据分析表明，经NMP处理的细菌细胞中，大量参与包膜应激响应的基因呈现差异表达特征。包膜应激响应与修复系统相关编码基因的上调，反映出细菌在暴露于亚抑菌浓度NMP的环境中生存时，其膜稳态遭到显著破坏。此外，编码自体诱导物-2（autoinducer-2）、参与生物膜形成的lsr操纵子出现表达下调，导致经NMP处理的细胞生物膜形成能力显著降低，该表型通过结晶紫染色实验得到了验证。本研究推测，早期发生的膜破坏会引发内膜电位去稳定化，进而损伤三磷酸腺苷（ATP）的生成过程，最终导致细菌外排泵功能被抑制。