Functional characterization of beta-lactam sensor proteins in Staphylococcus aureus

ANTINORI, M.A.; MIHOVILCEVIC, D.; FABBRI, C.; SUAREZ, I.P.; MÉNDEZ, L.; TESTERO, S.A.; LLARRULL, L.I.

Congreso; 20th IUPAB Congress, 50th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology (SBBq), 45th Congress of 50th Brazilian Society of Biophysics (SBBf) and 13th SBBn Congress; 2021

Staphylococcus aureus is the main cause of intra- and extra-hospital infections and is considered a high-priority multi-resistant pathogen. The manifestation of resistance to beta-lactam antibiotics in S. aureus is regulated by the transmembrane proteins BlaR1, MecR1 and VraS/VraT of the bla, mec and VraSRT systems. These proteins have extra-cytoplasmic domains that bind the antibiotic (BlaR1 and MecR1) or that might sense it (VraS and VraT). In this study we aim at unveiling the molecular details of the conformational change triggered by beta-lactams to activate the metalloprotease domain of BlaR1 and MecR1, and elucidating the mechanism of activation of VraS by beta-lactams. We combined the use of ampicillin-derived photoprobes, reporter strains, recombinant protein expression, phosphorylation assays, western blot, band-shift assays and electron microscopy. Using the S. aureus reporter strains we observed constitutive activation of the system for BlaR1-MN8 in contrast to inducible activation for wild type BlaR1, lower activation of the mec operon, and we confirmed that our ampicillin-derived photoprobes activate the VraSRT system. Incubation of recombinant VraS in E. coli spheroplasts with the activated photoprobes showed a band shift of VraS indicative of formation of a covalent adduct with the antibiotic, and an increase in VraS autokinase activity. Electron microscopy images of negatively stained VraS samples suggested formation of trimers or tetramers. We concluded that the mutation found in BlaR1-MN8 yields a constitutively-active metalloprotease, but with a lower activity than WT BlaR1, in agreement with the lower beta-lactamase activity seen in S. aureus MN8 in comparison with strain NRS128. Regarding the mec operon, we concluded that the level of expression mediated by β-lactam-activated MecR1 is significantly lower than that mediated by BlaR1, even upon expression of MecR2. In addition, our results suggested a direct interaction of beta-lactams with VraS, at a site yet to be elucidated, that upregulates autophosphorylation.