IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Characterization of the topology of the sensor/transducer proteins of the beta-lactam resistance systems from Methicillin-Resistant Staphylococcus aureus
Autor/es:
BELLUZO, B.S.; PERIS, D.; LLARRULL, L.I.*
Lugar:
Gran Canaria, Islas Canarias
Reunión:
Workshop; 12th beta-lactamase Meeting (International Workshop); 2014
Resumen:
The membrane proteins BlaR1 and MecR1 play key roles in resistance to β-lactam antibiotics in Methicillin-Resistant Staphylococcus aureus (MRSA) strains. It is proposed that they sense and transduce the signal to unleash the transcription of the resistance genes: the blaZ gene that codes for the S. aureus β-lactamase and the mecA gene that codes for the transpeptidase PBP2a that is capable of carrying out this key reaction in the synthesis of the peptidoglycan in the presence of otherwise inhibiting concentrations of β-lactams.1 The molecular events that are involved in the transmission of the signal remain controversial. Here we report a study of the topology of BlaR1 and MecR1 expressed in E. coli (where we have previously shown that BlaR1 is active)2 by means of the construction of C-terminal fusions to EGFP.3 This reporter does not fold correctly when translocated to the periplasmic space through the Sec System and, therefore, it does not fluoresce when fused to a periplasmic loop. In order to determine the number of membrane-spanning domains and the localization of the different domains, we characterized the different fusion proteins by fluorescence spectroscopy in whole cells and by fluorescence microscopy. The expression of the different constructions in E.coli was normalized by Western blot analysis. We used mutants on the Sec and Tat systems to verify the identity of the translocation system involved. This strategy allowed us to confirm that both BlaR1 and MecR1 adopt a similar topology, with the sensor domain exposed to the periplasmic space and the metalloprotease domain facing the cytoplasm in E. coli. The different fusion proteins accumulate in the membrane, and the full proteins BlaR1 and MecR1 in E. coli display an active sensor domain that could be labeled by a fluorescent β-lactam in whole cells. Acknowledgements The PEW Charitable Trusts, ANPCyT, CONICET References 1- Llarrull, L.I., Fisher, J.F., Mobashery, S. Antimicrob. Agents and Chemother. 2009, 53(10): 4051-63. 2- Llarrull, L.I., Mobashery, S. Biochemistry 2012, 51(23): 4642-9. 3- McKenzie, N.L., Nodwell, J.R. Microbiology. 2009,155, 1812-18.