金葡菌主动外排蛋白QacA α-螺旋1、2及TMS1氨基酸的功能研究

Functional study on amino acid residues in α-helix 1,2 and TMS1 of multidrug exporter QacA from Staphylococcus aureus

目的:评价金黄色葡萄球菌多药耐药主动外排蛋白Qac Aα-螺旋1和2及跨膜区(TMS)1上的氨基酸对于底物转运的重要性。方法:采用半胱氨酸定点诱变的方法诱变Qac A蛋白α-螺旋1和2及TMS1上的氨基酸I2、S3、F4、F5、T6、K7、D10、M11、R17、W18、T_19、A20、V21、V22、V23、F29、D34、P43、E48、E50、P51和Q55。测定诱变菌株最低抑菌浓度(MIC)和最低杀菌浓度(MBC)以及荧光运输试验分析氨基酸的功能变化。结果:MIC和MBC结果分析提示获得的诱变菌株S3C、T6C、R17C、T_19C、A20C、F29C和E50C对所测药物完全恢复敏感性,F4C、F5C和P51C对所测药物耐药性明显降低。运输试验结果与MIC、MBC结果一致,提示10株诱变菌株失去对底物的转运能力。结论:Qac A蛋白α-螺旋1和2及TMS1上的氨基酸S3、F4、F5、T6、R17、T_19、A20、F29、E50和P51可能与Qac A蛋白一价和二价阳离子底物的结合和转运功能密切相关。

AIM： To determine the importance of the amino acid residues in α-helix 1,2 and transmembrane segment（ TMS） 1 of multidrug exporter Qac A from Staphylococcus aureus. METHODS： Cysteine site-directed mutagenesis was used to mutate the residues including I2,S3,F4,F5,T6,K7,D10,M11,R17,W18,T19,A20,V21,V22,V23,F29,D34,P43,E48,E50,P51 and Q55. Minimum inhibitory concentration（ MIC） and minimum bactericidal concentration（ MBC） determination and transport assay were performed to analyze the function of these mutants. RESULTS： The results of MIC and MBC analysis suggested that the drug sensitivity of the mutants S3 C,T6C,R17 C,T19C,A20 C,F29C and E50 C to the detected substrates restored completely,and the resistance of the mutants F4 C,F5C and P51 C to the detected substrates reduced significantly. The results of the fluorimetric transport assay were in agreement with those of MIC and MBC analysis,which indicated that the mutants of S3 C,F4C,F5 C,T6C,R17 C,T19C,A20 C,F29C,E50 C and P51 C could not transport the tested substrates. CONCLUSION： The residues including S3,F4,F5,T6,R17,T19,A20,F29,E50 and P51 are probably involved in the substrate binding and translocation in α-helix 1,2 and TMS1 of multidrug exporter Qac A.