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，具有内在和多重耐药性。这种细菌产生抗生素耐药性的主要原因似乎是在mex操纵子（mexa - mex13 - oprm）中编码了一种能量依赖的抗生素挤出泵（MexAB-OprM泵）。为了深入了解Mex泵对抗生素的挤出作用模式，有必要对其各亚基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，认为MexA将内膜泵MexB与外膜通道OprM连接起来。我使用基因融合技术分析了MexA的膜拓扑结构，使用无信号序列的β -内酰胺酶作为报告基因，得到了78个帧内的MexA- β -内酰胺酶融合。携带每个融合基因的所有转化子都表现出氨苄西林抗性，这表明几乎所有MexA信号序列以外的区域都位于外质。在脂蛋白的一致序列中，还发现MexA的半胱氨酸残基被脂质修饰。

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.