Role of TMS5: staphylococcal multidrug-efflux protein QacA.
BACKGROUND
QacA, a main exporter mediating the multidrug-resistance of Staphylococcus aureus to a variety of antiseptics and disinfectants, possesses a topology of 14 alpha-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 fluorimetric 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.
Jia
Bei
B
Key Laboratory of Infectious and Parasitic Diseases of Chongqing, Infectious Disease Department, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China. beijia123@sina.com
Zhou
Ting-quan
TQ
Huang
Ai-long
AL
Huang
Wen-xiang
WX
D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D024881:Drug Resistance, Bacterial; D005033:Ethylmaleimide / Q000494:pharmacology; D007211:Indoles / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000502:physiology*; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D007211:Indoles; D026901:Membrane Transport Proteins; C068498:qacA protein, Staphylococcus aureus; C007293:DAPI; D005033:Ethylmaleimide