Role of TMS5： staphylococcal multidrug-efflux protein QacA  

作　　者：JIA Bei ZHOU Ting-quan HUANG Ai-long HUANG Wen-xiang 

机构地区：[1]Key Laboratory of Infectious and Parasitic Diseases of Chongqing, Infectious Disease Department, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China [2]Key Laboratory of Molecular Biology on Infectious Diseases of the Education Ministry, Chongqing Medical University Institute of Viral Hepatitis, Secondary Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China

出　　处：《Chinese Medical Journal》2008年第5期409-413,共5页中华医学杂志（英文版）

基　　金：This project was supported by a grant from National Natural Science Foundation of China （No. 30400373）.

摘　　要：Background QacA, a main exporter mediating the multidrug-resistance of Staphylococcus aureus to a vanety of antiseptics and disinfectants, possesses a topology of 14 a-helical transmembrane segments （TMS）. Our study aimed to determine the importance and topology of amino acid residues in and flanking the cytoplasmic end of TMS5. Methods Site-directed mutagenesis was used to mutate 5 residues, including L146, A147, V148, W149 and S150, into cysteine. A minimum inhibitory concentration （MIC） and transport assay with or without N-ethylmaleimide （NEM） were performed to analyse the function of these mutants. Results All of the mutants showed comparable protein expression levels. MIC analysis suggested that mutant W149C showed low resistance levels to the drugs, but the mutations at L146, A147, V148, and S150C had little or no effect on the resistance level. And the results of the fluorimetdc transport assay were in agreement with those of MIC analysis, that is to say, W149C did not allow transport to the substrates to be tested, while the other mutants retained significant transport ability. The reaction of the different mutant proteins with Fluorescein-NEM revealed that the mutant L146C was highly reactive with NEM; the W149C and S150C mutants were moderately reactive; A147C was barely reactive and V148C showed no reactivity. Conclusions The study identified that residues W149 and S150 situated at the interface of the aqueous： lipid junction as functionally important residues, probably involved in the substrate binding and translocation of QacA.Background QacA, a main exporter mediating the multidrug-resistance of Staphylococcus aureus to a vanety of antiseptics and disinfectants, possesses a topology of 14 a-helical transmembrane segments （TMS）. Our study aimed to determine the importance and topology of amino acid residues in and flanking the cytoplasmic end of TMS5. Methods Site-directed mutagenesis was used to mutate 5 residues, including L146, A147, V148, W149 and S150, into cysteine. A minimum inhibitory concentration （MIC） and transport assay with or without N-ethylmaleimide （NEM） were performed to analyse the function of these mutants. Results All of the mutants showed comparable protein expression levels. MIC analysis suggested that mutant W149C showed low resistance levels to the drugs, but the mutations at L146, A147, V148, and S150C had little or no effect on the resistance level. And the results of the fluorimetdc transport assay were in agreement with those of MIC analysis, that is to say, W149C did not allow transport to the substrates to be tested, while the other mutants retained significant transport ability. The reaction of the different mutant proteins with Fluorescein-NEM revealed that the mutant L146C was highly reactive with NEM; the W149C and S150C mutants were moderately reactive; A147C was barely reactive and V148C showed no reactivity. Conclusions The study identified that residues W149 and S150 situated at the interface of the aqueous： lipid junction as functionally important residues, probably involved in the substrate binding and translocation of QacA.

关 键 词：MULTIDRUG-RESISTANT staphylococcus aureus EXPORTER QacA TMS5 

分 类 号：R969.3[医药卫生—药理学]