PDF(Pubmed)
Abstract：
Multidrug efflux pumps are the frontline defense mechanisms of Gram-negative bacteria, yet little is known of their relative fitness trade-offs under gut conditions such as low pH and the presence of antimicrobial food molecules. Low pH contributes to the proton-motive force (PMF) that drives most efflux pumps. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. Competition assays were performed by flow cytometry of co-cultured Escherichia coli K-12 strains possessing or lacking a given pump complex. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with carbonyl cyanide 3-chlorophenylhydrazone or at pH 8.0). At pH 5.5, selection against AcrAB-TolC was increased by aromatic acids, alcohols, and related phytochemicals such as methyl salicylate. The degree of fitness cost for AcrA was correlated with the phytochemical\'s lipophilicity (logP). Methyl salicylate and salicylamide selected strongly against AcrA, without genetic induction of drug resistance regulons. MdtEF-TolC and EmrAB-TolC each had a fitness cost at pH 5.5, but salicylate or benzoate made the fitness contribution positive. Pump fitness effects were not explained by gene expression (measured by digital PCR). Between pH 5.5 and 8.0, acrA and emrA were upregulated in the log phase, whereas mdtE expression was upregulated in the transition-to-stationary phase and at pH 5.5 in the log phase. Methyl salicylate did not affect pump gene expression. Our results suggest that lipophilic non-acidic molecules select against a major efflux pump without inducing antibiotic resistance regulons.IMPORTANCEFor drugs that are administered orally, we need to understand how ingested phytochemicals modulate drug resistance in our gut microbiome. Bacteria maintain low-level resistance by proton-motive force (PMF)-driven pumps that efflux many different antibiotics and cell waste products. These pumps play a key role in bacterial defense by conferring resistance to antimicrobial agents at first exposure while providing time for a pathogen to evolve resistance to higher levels of the antibiotic exposed. Nevertheless, efflux pumps confer energetic costs due to gene expression and pump energy expense. The bacterial PMF includes the transmembrane pH difference (ΔpH), which may be depleted by permeant acids and membrane disruptors. Understanding the fitness costs of efflux pumps may enable us to develop resistance breakers, that is, molecules that work together with antibiotics to potentiate their effect. Non-acidic aromatic molecules have the advantage that they avoid the Mar-dependent induction of regulons conferring other forms of drug resistance. We show that different pumps have distinct selection criteria, and we identified non-acidic aromatic molecules as promising candidates for drug resistance breakers.
摘要：
多药外排泵是革兰氏阴性菌的前线防御机制，然而，在肠道条件下，如低pH和抗微生物食品分子的存在，他们的相对适应性权衡知之甚少。低pH有助于驱动大多数外排泵的质子动力(PMF)。我们展示了依赖PMF的泵AcrAB-TolC，MdtEF-TolC,和EmrAB-TolC在低pH值和膜渗透性植物化学物质存在下进行选择。通过流式细胞术对具有或缺乏给定泵复合物的共培养的大肠杆菌K-12菌株进行竞争测定。在耗尽PMF的条件下(用羰基氰化物3-氯苯腙pH5.5或pH8.0),所有三个泵均显示阴性选择。在pH5.5时，芳香酸增加了对AcrAB-TolC的选择，酒精,和相关的植物化学物质，如水杨酸甲酯。AcrA的适应成本程度与植物化学物质的亲脂性（logP）相关。水杨酸甲酯和水杨酰胺强烈对抗AcrA，没有遗传诱导的耐药性调节子。MdtEF-TolC和EmrAB-TolC在pH5.5时各自具有健身成本，但水杨酸盐或苯甲酸盐对健身的贡献为正。泵适应性影响不能通过基因表达（通过数字PCR测量）来解释。在pH5.5和8.0之间，acrA和emrA在对数阶段上调，而mdtE表达在过渡到固定阶段和在对数阶段的pH5.5上调。水杨酸甲酯不影响泵基因表达。我们的结果表明，亲脂性非酸性分子选择主要的外排泵而不会诱导抗生素抗性调节子。重要性对于口服给药的药物，我们需要了解摄入的植物化学物质如何调节肠道微生物组的耐药性。细菌通过质子动力（PMF）驱动的泵保持低水平的抵抗力，这些泵流出许多不同的抗生素和细胞废物。这些泵通过在首次暴露时赋予抗微生物剂抗性而在细菌防御中起关键作用,同时为病原体提供时间以进化对更高水平的暴露抗生素的抗性。然而，外排泵由于基因表达和泵能量费用而产生能量成本。细菌PMF包括跨膜pH差(ΔpH)，可能被渗透酸和膜破坏物耗尽。了解外排泵的健身成本可能使我们能够开发阻力断路器，也就是说,与抗生素一起工作以增强其作用的分子。非酸性芳族分子的优点在于它们避免了赋予其他形式的耐药性的调节子的Mar依赖性诱导。我们表明不同的泵有不同的选择标准，我们确定了非酸性芳香分子是有希望的候选药物的耐药性破坏剂。
