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菌，具有内在耐药性和多重耐药性。该细菌中抗生素抗性的主要原因似乎是存在编码在mex操纵子（mexA-mexl 3-oprM）中的能量依赖性抗生素挤出泵（MexAB-OprM泵）。为了深入了解Mex泵对抗生素的挤出方式，有必要对Mex泵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，该蛋白被认为连接内膜泵MexB和外膜通道OprM。我采用基因融合技术，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个符合读框的MexA-β-内酰胺酶融合体。每个融合基因的所有转化体都表现出氨苄青霉素抗性，这表明几乎所有的MexA区域都位于周质中。还发现MexA在脂蛋白共有序列中的半胱氨酸残基处被脂质修饰。

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.