Unit of Mycobacteriology, Universidade Nova de Lisboa (IHMT/UNL), Lisboa, Portugal.
PLoS One. 2009 Aug 17;4(8):e6656. doi: 10.1371/journal.pone.0006656.
Resistance Nodulation Division (RND) efflux pumps of Escherichia coli extrude antibiotics and toxic substances before they reach their intended targets. Whereas these pumps obtain their energy directly from the proton motive force (PMF), ATP-Binding Cassette (ABC) transporters, which can also extrude antibiotics, obtain energy from the hydrolysis of ATP. Because E. coli must pass through two pH distinct environments of the gastrointestinal system of the host, it must be able to extrude toxic agents at very acidic and at near neutral pH (bile salts in duodenum and colon for example). The herein described study examines the effect of pH on the extrusion of ethidium bromide (EB).
METHODOLOGY/PRINCIPAL FINDINGS: E. coli AG100 and its tetracycline induced progeny AG100(TET) that over-expresses the acrAB efflux pump were evaluated for their ability to extrude EB at pH 5 and 8, by our recently developed semi-automated fluorometric method. At pH 5 the organism extrudes EB without the need for metabolic energy (glucose), whereas at pH 8 extrusion of EB is dependent upon metabolic energy. Phe-Arg beta-naphtylamide (PAbetaN), a commonly assumed inhibitor of RND efflux pumps has no effect on the extrusion of EB as others claim. However, it does cause accumulation of EB. Competition between EB and PAbetaN was demonstrated and suggested that PAbetaN was preferentially extruded. A K(m) representing competition between PAbetaN and EB has been calculated.
CONCLUSIONS/SIGNIFICANCE: The results suggest that E. coli has two general efflux systems (not to be confused with a distinct efflux pump) that are activated at low and high pH, respectively, and that the one at high pH is probably a putative ABC transporter coded by msbA, which has significant homology to the ABC transporter coded by efrAB of Enterococcus faecalis, an organism that faces similar challenges as it makes its way through the toxic intestinal system of the host.
大肠杆菌的抗性调节与分解代谢基因群（RND）外排泵在抗生素和有毒物质到达靶标之前将其排出。虽然这些泵直接从质子动力势（PMF）中获取能量，但也能排出抗生素的三磷酸腺苷结合盒（ABC）转运蛋白则从 ATP 的水解中获取能量。由于大肠杆菌必须通过宿主胃肠道系统的两个 pH 值截然不同的环境，因此它必须能够在非常酸性和接近中性 pH 值（例如十二指肠和结肠中的胆汁盐）下排出有毒物质。本研究探讨了 pH 值对溴化乙锭（EB）排出的影响。
方法/主要发现：使用我们最近开发的半自动化荧光法，评估了大肠杆菌 AG100 及其四环素诱导的后代 AG100（TET）在 pH 值为 5 和 8 时排出 EB 的能力。在 pH 值为 5 时，该生物体在不需要代谢能量（葡萄糖）的情况下排出 EB，而在 pH 值为 8 时，EB 的排出依赖于代谢能量。苯丙氨酸-精氨酸-β-萘基酰胺（PAbetaN），一种通常被认为是 RND 外排泵抑制剂的物质，如其他人所声称的那样，对 EB 的排出没有影响。然而，它确实会导致 EB 的积累。已经证明了 EB 和 PAbetaN 之间的竞争，并表明 PAbetaN 被优先排出。已经计算出代表 PAbetaN 和 EB 之间竞争的 K（m）。
结论/意义：结果表明，大肠杆菌有两种一般的外排系统（不要与独特的外排泵混淆），分别在低 pH 值和高 pH 值下激活，而高 pH 值下的系统可能是一种假定的 ABC 转运蛋白，由 msbA 编码，与粪肠球菌的 efrAB 编码的 ABC 转运蛋白具有显著同源性，粪肠球菌在其通过宿主毒性肠道系统的过程中面临类似的挑战。
