Studies in membrane topology and function of inner membrane component, MexA,of antibiotic extrusion pump of Pseudomonas aeruginosa

铜绿假单胞菌是机会性感染的主要病原体，表现出内在的多重抗生素耐药性。这种细菌产生耐药性的主要原因可能是存在一个能量依赖的抗生素挤出泵(MexAB-OprM泵)，该泵编码在Mex操纵子(MexA-mexl3-oprM)中。为了深入了解MEX泵挤出抗生素的方式，有必要对泵的各个亚单位的结构和功能进行研究。因此，我分析了膜融合蛋白家族成员MexA的膜拓扑结构和定位，MexA被认为是连接内膜泵MexB和外膜通道OprM的成员。我利用基因融合技术，以无信号序列的β-内酰胺酶为报告基因，分析了MexA的膜拓扑结构，获得了78个框内MexA-β-内酰胺酶融合。所有含有每个融合基因的转化子都表现出对氨苄西林的抗性，这表明MexA信号序列以外的几乎所有区域都位于周质中。还发现MexA在脂蛋白共有序列的半胱氨酸残基上被脂质(S)修饰。

Pseudomonas aeruginosa, a main causative agent of opportunistic infections, shows intrinsic and multiantibiotic resistance. The major reason for the antibiotic resistance in this bacterium appeared to be the presence of an energy dependent antibiotic extrusion pump (MexAB-OprM pump) encoded in the mex operon (mexA-mexl3-oprM). To get insight into the mode of antibiotic extrusion by the Mex pump, it is essential to study on the structure and function of each subunit of the pump. Therefore, I analyzed the membrane topology and localization of MexA, a member of membrane fusion protein family, which is thought to link inner membrane pump, MexB, to an outer membrane channel, OprM.I employed the gene fusion technique to analyze the membrane topology of MexA using signal sequenceless beta-lactamase as a reporter and got 78 in-frame MexA-beta--lactamase fusions. All transformants harbored each fusion gene showed ampicillin resistance, indicating that almost all regions of MexA beyond the signal sequence reside in the periplasm. MexA was also found to be modified by lipid(s) at its cysteine residue in the consensus sequence for lipoproteins.