VbrKR, a b-lactam resistance system in gram negative bacteria

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

Rosario

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

Sociedad Argentina de Biofísica

Emergence of b-lactam resistant bacteria represents a public health challenge worldwide. Resistant strains of the Vibrio genus (causing agent of acute intestinal infections) have been reported around the world, constituting a threat to human health, especially in developing countries. A b-lactam resistance system has been recently described in Vibrio parahaemolyticus. The resistance is activated by a two-component system consisting of a membrane histidine kinase (VbrK) and a cytoplasmic response regulator (VbrR). In the presence of b-lactams, the system activates the expression of a CARB b-lactamase. Our goal is to understand the molecular mechanism underlying 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 b-lactam molecule, but this still remains controversial. To study the recognition of b-lactams by VbrK we have optimized the expression and purification of its sensor domain (VbrKS). We have confirmed the predicted cleavage site of the signal peptide by Mass-spectrometry. Combining fluorescence spectroscopy, thermal shift assays, Ellman’s reagent and NMR we describe the interplay between the domain’s stability, the oxidation state of the cysteine residues, and its affinity for different b-lactams. To identify the binding region, we have developed a photoprobe derived from ampicillin (Dz-Amp). This reagent contains a photoactive diazirine group which upon UV irradiation generates a carbene that reacts with surrounding aminoacidic sidechains creating a covalent link. On the other hand, we have included a F3C-group in the molecule that allows monitoring the reaction by 19F-NMR. We have employed Dz-Amp to covalently modify VbrKS and identified the modified protein by MS and 19F-NMR. Our results suggest that VbrKS directly binds b-lactams.