Regulation of β-Lactamase Synthesis as a Novel Site of Action for Suppression of Methicillin Resistance in Staphylococcus aureus

Abstract

Nearly all clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) produce beta-lactamase as well as an additional low-affinity penicillin-binding protein called PBP2a or PBP2', the main factor for mediating methicillin resistance. Polidocanol (PDO), a dodecyl polyethyleneoxide ether, resensitizes clinical isolates of MRSA to methicillin; in addition, their resistance to benzylpenicillin (BP) is reduced. The action of PDO is based on the inhibition of the induced syntheses of PBP2a and beta-lactamase. Induction in our study was performed with 2-(2'-carboxyphenyl)benzoyl-6-aminopenicillanic acid (CBAP). Inducible PBP2a production in MRSA strains is under the control of the same regulatory system which is responsible for the induction of beta-lactamase synthesis. BlaR1, a membrane-spanning protein with a penicillin sensor and a signal transducer domain represents the starting point of this induction cascade. Based on its amphiphilic properties, it is likely that the action of PDO is located in the bacterial membrane. Therefore we investigated the possibility that BlaR1 might be the main target for PDO action. We were able to detect the BlaR1 sensor domain in resistant staphylococcal cells even in the noninduced state by fluorography. In a competition assay, CBAP was bound specifically, with a high affinity to the penicillin sensor. Moreover, the binding of CBAP was very stable. As concerns PDO, no significant interaction with the penicillin binding site of BlaR1 was detectable. This is why the BlaR1 transducer domain is thought to be the actual target area of PDO. In this case, PDO would interfere with the transmission of the signal, generated by the receptor binding of CBAP, through the membrane via BlaR1 into the staphylococcal cell. This assumption could be confirmed by the analysis of the concentration-effect relationship, whereafter PDO does not work as a competitive, but as a noncompetitive antagonist of CBAP. Our results demonstrate that BlaR1 could be an attractive new target for the development of new drugs to overcome methicillin resistance.