基于鲍曼不动杆菌AdeABC分子的新型杂合外排泵模型的构建与评价

Multidrug-resistant Acinetobacter baumannii is becoming an increasingly serious problem worldwidely, and the main resistance mechanism is tightly associated with the overexpression of the multidrug efflux transporter AdeABC, which leads to expel antibiotic out of the bacterial membrane. However, there is still no effective efflux pump inhibitors (EPIs) to block-up the AdeABC transporter because of the shortcomings of low effectiveness and high interference for the now available screening model for EPIs. Further bioimformatics analysis reveal that the AdeABC transporter from A. baumannii and the AcrAB-TolC molecules from Escherichia coli is highly conserved in sequence and structure, because both systems belong to the resistance-nudolation-division (RND) family which efflux substrate through the double menbrane of Gram-negative bacterium by 3 different subunits coordinatly. S The major goal of the present study is to duplicate the efflux system of A. baumannii in non-drug-resistant E. coli strain W3110 by expressing the antibiotics-capture subunit AdeB and the effluxing subunit AdeC from A. baumannii in a knockout mutant E. coli of which the isogenic subunit of AcrB and TolC has been knocked out by our group. So a heterozygous efflux pump AcrAB-TolC is generated, and the function and characteristics of this efflux will be tested and verified. The newly established heterozygous pump model can be examined as a whole to screen EPIs candidates, and prevent the interference of other resistance mechanisms of A. baumannii, as well as to remedy the inadequacy of the available EPIs screening models. Therefore, it would provide one more efficacious model for screening EPIs against the AdeABC transporter, thus facilitating drug design and discovery for antibacterial therapy of multidrug-resistant A. baumannii.

鲍曼不动杆菌的多耐药性问题日趋严重，其原因与该菌外膜上外排泵AdeABC分子的过表达导致抗生素外流密切相关。但至今尚无针对AdeABC的有效抑制剂，这与现有外排泵抑制剂筛选模型存在高干扰性和低有效性等有关。进一步分析发现，鲍曼不动杆菌AdeABC与大肠杆菌AcrAB-TolC同为耐药结节分生家族，且生物信息学结果显示两个不同来源的外排泵分子在结构与功能上高度保守。因此，本研究拟在已构建大肠杆菌W3110株的acrB和tolC基因敲除株的基础上，将鲍曼不动杆菌相应的抗生素捕获分子adeB及外排分子adeC转化入该敲除菌中表达，旨在利用无耐药性大肠杆菌复制出具有鲍曼不动杆菌外排特性的杂合外排泵AcrA-AdeBC，并评价该杂合外排泵的活性及特性。该新型杂合外排泵以完整的外排泵为筛选靶点，可排除其他耐药因素的干扰并克服现有筛选系统的不足，为鲍曼不动杆菌AdeABC外排泵抑制剂的筛选提供有效模型。

鲍曼不动杆菌的多耐药性问题日趋严重，其原因与该菌外膜上外排泵AdeABC分子的过表达导致抗生素外流密切相关。但至今尚无针对AdeABC的有效抑制剂，这与现有外排泵抑制剂筛选模型存在高干扰性和低有效性等有关。进一步分析发现，鲍曼不动杆菌AdeABC与大肠杆菌AcrAB-TolC同为耐药结节分生家族，且生物信息学结果显示两个不同来源的外排泵分子在结构与功能上高度保守。本研究首先构建大肠杆菌W3110株的acrB和tolC基因敲除株，随后将鲍曼不动杆菌外排泵AdeABC中的外排分子adeC转化入该敲除菌中表达，成功地在无耐药性大肠杆菌复制出具有鲍曼不动杆菌外排特性的杂合外排泵AcrAB-AdeC，进一步验证该杂合外排泵具有鲍曼不动杆菌的抗性特征。该新型杂合外排泵在大肠杆菌中表达且具有鲍曼不动杆菌外排特性，可作为鲍曼不动杆菌AdeABC外排泵抑制剂的筛选候选模型。