Absence, loss-of-function, or inhibition of Escherichia coli AcrB does not increase expression of other efflux pump genes supporting the discovery of AcrB inhibitors as antibiotic adjuvants.
Absence, loss-of-function, or inhibition of Escherichia coli AcrB does not increase expression of other efflux pump genes supporting the discovery of AcrB inhibitors as antibiotic adjuvants.
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DOI：
10.1093/jac/dkab452
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发表时间：
2022-02-23
期刊：
影响因子：
--
通讯作者：
Piddock LJV
中科院分区：
文献类型：
--
作者：
Ciusa ML;Marshall RL;Ricci V;Stone JW;Piddock LJV
确定是否外排泵的表达和抗生素敏感性在大肠杆菌中改变，以响应外排抑制。将9个外排泵基因（acrAB、acrD、acrEF、emrAB、macAB、cusCFBA、mdtK、mdtABC、mdfA）的启动子区与pMW 82中的gfp融合，并且将来自每个报告构建体的荧光用作对AcrB被抑制、缺失或变得无功能的条件的转录应答的量度。还通过RT-qPCR测定表达。研究了具有已知或预测会导致编码外排泵功能丧失的错义突变的外排泵突变体的药物敏感性。来自GFP报告基因构建体的数据显示，当AcrB不存在、无功能或被外排泵抑制剂/竞争性底物（如PAβN或氯丙嗪）抑制时，未观察到测试的外排泵基因的表达增加。PAβN和氯丙嗪的RT-qPCR证实了这一点;然而，当acrB缺失时，观察到macB基因表达小幅但显著增加。外排抑制剂仅在功能性AcrB存在下与抗生素协同作用。当AcrB不存在或无功能时，当其他外排泵也无功能时，对MIC没有影响。大肠杆菌缺失、功能丧失或抑制。coli AcrB没有显著增加其他外排泵基因的表达，这表明没有补偿机制来克服外排抑制，并支持发现AcrB抑制剂作为抗生素佐剂。
To determine whether expression of efflux pumps and antibiotic susceptibility are altered in Escherichia coli in response to efflux inhibition. The promoter regions of nine efflux pump genes (acrAB, acrD, acrEF, emrAB, macAB, cusCFBA, mdtK, mdtABC, mdfA) were fused to gfp in pMW82 and fluorescence from each reporter construct was used as a measure of the transcriptional response to conditions in which AcrB was inhibited, absent or made non-functional. Expression was also determined by RT-qPCR. Drug susceptibility of efflux pump mutants with missense mutations known or predicted to cause loss of function of the encoded efflux pump was investigated. Data from the GFP reporter constructs revealed that no increased expression of the tested efflux pump genes was observed when AcrB was absent, made non-functional, or inhibited by an efflux pump inhibitor/competitive substrate, such as PAβN or chlorpromazine. This was confirmed by RT-qPCR for PAβN and chlorpromazine; however, a small but significant increase in macB gene expression was seen when acrB is deleted. Efflux inhibitors only synergized with antibiotics in the presence of a functional AcrB. When AcrB was absent or non-functional, there was no impact on MICs when other efflux pumps were also made non-functional. Absence, loss-of-function, or inhibition of E. coli AcrB did not significantly increase expression of other efflux pump genes, which suggests there is no compensatory mechanism to overcome efflux inhibition and supports the discovery of inhibitors of AcrB as antibiotic adjuvants.
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影响因子：
3.7
作者：
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通讯作者：
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作者：
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DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
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通讯作者：
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