CONICET | Buscador de Institutos y Recursos Humanos

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a globally important pathogen that is resistant to all classes of β-lactam antibiotics and some strains also are resistant to glycopeptides, the last resort antibiotics used to treat MRSA infections. Resistance to β-lactam antibiotics in MRSA is due to the inducible expression of an accessory transpeptidase, PBP2a, with low affinity for most β-lactams and of the serin-β-lactamase PC1. The sensor/transducer proteins MecR1 and BlaR1 regulate the level of expression of PBP2a and PC1, respectively, in response to the presence of the β-lactam antibiotic. Little is known about the intramolecular events that lead to activation of BlaR1 and MecR1, and the search for inhibitors has been limited by the lack of high resolution structural information on the full-length proteins.In order to gain insight into the molecular details of the activation of BlaR1 and MecR1 we have evaluated the expression of full-length BlaR1 and MecR1 and also of shorter versions of these two proteins (with fewer transmembrane helixes) as fusions to Mistic. We succeeded in overexpressing Mistic-BlaR1-JH1 in E. coli BL21 StarTM (DE3) membranes and we were able to purify it to homogeneity using affinity chromatography. We conducted Proteinase K susceptibility assays in spheroplasts to evaluate the topology of BlaR1-JH1 when fused to Mistic. We also verified the extracellular localization of the sensor domain of BlaR1-JH1 in this construct by incubation of spheroplasts with the fluorescent antibiotic Bocillin-FL. We expressed MecR1-E205A (no auto-proteolytic activity) with a C-terminal His-6x tag in E. coli BL21 StarTM (DE3). However, MecR1-E205A was still prone to proteolysis by native E. coli proteases. In order to identify the later site of proteolysis by MALDI-TOF/TOF, we purified MecR1 from membranes by affinity chromatography for analysis of the full-length protein and of the proteolysis bands by MALDI-TOF/TOF.