Beta-lactam recognition by VbrK kinase from V. parahaemolyticus

PALANCA, I.; SUAREZ, I.P.; LACAVA, F.; MÉNDEZ, L.; TESTERO, S.A.; LLARRULL, L.I.

Tandil

Taller; V Taller Argentino de Resonancia Magnética; 2023

Asociación Física Argentina

Emergence of β-lactam resistant bacteria represents a public health challenge worldwide. Resistantstrains of the Vibrio genus (causing agent of acute intestinal infections) have been reported aroundthe world, constituting a threat to human health, especially in developing countries. A β-lactam antibiotic resistance system has been recently described in Vibrio parahaemolyticus. The esistance is activated by a two-component system consisting of a membrane histidine kinase (VbrK) and a cytoplasmic response regulator (VbrR). Upon β-lactam presence the system activates the expression of a CARB β-lactamase. Our goal is to understand the molecular mechanism underlying the antibiotic detection and signal transduction resulting in the resistance.VbrK consists of a signal peptide, a putative periplasmic sensor domain, a transmembrane helix, and cytoplasmic histidine kinase and ATPase domains. The signal detected by the kinase is proposed to be the β-lactam molecule, but this still remains controversial. To study the recognition of β-lactams by VbrK we have optimized the expression and purification of its sensor domain (VbrKSD). We have confirmed the predicted cleavage site of the signal peptide by Mass-spectrometry. Combining fluorescence spectroscopy, Ellman’s assay and NMR we describe the interplay between the cysteine´s oxidation state, and its affinity for different β-lactams. To identify the binding region, we have synthetized two photoprobes derived from ampicillin, Dz-Amp and Az-Amp. These reagents contain a photoactivable diazirine or azide group respectively, which upon UV irradiation generate a free radical that reacts with surrounding aminoacidic sidechains creating a covalent link. On the other hand, we have included 19F atoms in the photoprobe molecules that allows monitoring the reaction by 19F-NMR. We have employed Dz-Amp to covalently modify VbrKSD and identified the modified protein by MS and 19F-NMR. Our results suggest that VbrKSD is directly binding β-lactams, and the binding site is not the same as suggested in previous literature. These results, in addition to bioinformatic analysis, contribute to elucidate the molecular mechanism of this sensor kinase.