School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
J Antimicrob Chemother. 2009 Nov;64(5):965-72. doi: 10.1093/jac/dkp311. Epub 2009 Sep 10.
AcrA can function as the periplasmic adaptor protein (PAP) in several RND tripartite efflux pumps, of which AcrAB-TolC is considered the most important. This system confers innate multiple antibiotic resistance. Disruption of acrB or tolC impairs the ability of Salmonella Typhimurium to colonize and persist in the host. The aim of this study was to investigate the role of AcrA alone in multidrug resistance and pathogenicity.
The acrA gene was inactivated in Salmonella Typhimurium SL1344 by insertion of the aph gene and this mutant complemented with pWKS30acrA. The antimicrobial susceptibility of the mutant to six antibiotics as well as various dyes and detergents was determined. In addition, efflux activity was quantified. The ability of the mutant to adhere to, and invade, tissue culture cells in vitro was measured.
Following disruption of acrA, RT-PCR and western blotting confirmed that acrB/AcrB was still expressed when acrA was disrupted. The acrA mutant was hypersusceptible to antibiotics, dyes and detergents. In some cases, lower MICs were seen than for the acrB or tolC mutants. Efflux of the fluorescent dye Hoechst H33342 was less than in wild-type following disruption of acrA. acrA was also required for adherence to, and invasion of, tissue culture cells.
Inactivation of acrA conferred a phenotype distinct to that of acrB::aph and tolC::aph. These data indicate a role for AcrA distinct to that of other protein partners in both efflux of substrates and virulence.
AcrA 可以作为几种 RND 三组分外排泵的周质适应蛋白（PAP），其中 AcrAB-TolC 被认为是最重要的。该系统赋予固有多重抗生素耐药性。破坏 acrB 或 tolC 会损害鼠伤寒沙门氏菌在宿主中定植和持续存在的能力。本研究旨在研究 AcrA 单独在多药耐药性和致病性中的作用。
通过插入 aph 基因，使鼠伤寒沙门氏菌 SL1344 中的 acrA 基因失活，并使用 pWKS30acrA 对该突变体进行互补。测定突变体对六种抗生素以及各种染料和去污剂的抗菌敏感性。此外，还定量了外排活性。测定突变体在体外黏附和侵袭组织培养细胞的能力。
在破坏 acrA 后，RT-PCR 和 Western blot 证实，当 acrA 被破坏时，acrB/AcrB 仍被表达。acrA 突变体对抗生素、染料和去污剂高度敏感。在某些情况下，与 acrB 或 tolC 突变体相比，MIC 更低。破坏 acrA 后，荧光染料 Hoechst H33342 的外排小于野生型。acrA 还需要黏附和侵袭组织培养细胞。
失活 acrA 赋予了不同于 acrB::aph 和 tolC::aph 的表型。这些数据表明，AcrA 在底物外排和毒力方面的作用与其他蛋白伴侣不同。
