Architecture of MecR1 of Staphylococcus aureus: clues to the signal transduction mechanism that unleashes resistance to β-lactams.

LLARRULL, L.I.; FABBRI, C.

Montevideo

Workshop; 4th Protein Biophysics at the end of the world; 2019

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 are also 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. However, the intramolecular events that lead to activation of BlaR1 and MecR1 are poorly understood, 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 MecR1 we have evaluated the expression of full-length MecR1 (E205A mutant, which lacks auto-proteolytic activity) as a fusion to the protein Mistic. We have succeeded in overexpressing Mistic-MecR1 in E. coli BL21 StarTM (DE3) membranes and we were able to purify it in detergent micelles to homogeneity using affinity chromatography. Activity assay of MecR1 sensor domain with the fluorescent antibiotic Bocillin-FL showed an active sensor domain in this recombinant protein. In addition, we have obtained polycrystals by crystallographic test, nevertheless, in a subsequent optimization, the formation of individual crystals has not been achieved yet. I am currently exploring our proposed mechanism of activation of MecR1 by β-lactams using site-directed mutagenesis in a S. aureus reporter strain, and using photoactive amino acids .