Functional characterization of the mec system and development of a crosslinking-strategy to study the signal transduction mechanism

FABBRI, C.; LLARRULL, L.I.

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

In methicillin-resistant Staphylococcus aureus, the mec system confers resistance to all classes of b-lactams antibiotics through the production of an accessory transpeptidase with low affinity for these antibiotics. The expression of PBP2a is inducible and it was historically proposed that, due to its structural homology, the mec system would function like the better characterized, bla operon. The intramolecular events that lead to the activation of MecR1/BlaR1 sensor proteins are yet to be elucidated.On one side, we studied the molecular events that give rise to the activation of the mec system using a S. aureus RN4220 reporter strain and RT-qPCR. 􀀁-Lactam-induced activation of transcription/translation did not result in manifestation of resistance. Nevertheless, we confirmed the inducible expression of PBP2a in membranes of the reporter strain through labeling with the fluorescent antibiotic Bocillin-FL and mass spectrometry. Based on published results, we hypothesize that the lipid composition of the S. aureus RN4220 membrane prevents activation of PBP2a. UV-Vis spectra of staphyloxanthins extracted from the reporter strain and clinical strains showed differences in the carotenoid content, which can explain the absence of resistance in the reporter strain. Overall, our results reveal significant differences in the mechanism of regulation ofthe resistance determinants by the bla and mec systems.On the other side, in order to unveil the conformational changes involved in activation of the sensor/transducer protein MecR1, we incorporated the unnatural photoactivatable amino acid p-azido-phenylalanine (pAzF). After expressing and purifying the irradiated mutant proteins in the presence and absence of antibiotic, they were subjected to in-gel trypsin digestion and analyzed by ESI-MS/MS to map intramolecular interactions. We found no evidence of intramolecular crosslinking, which poise a reevaluation of the crosslinking strategy and/or the proposed MecR1 model.