Redox-regulated cysteine acylation governs β-lactam sensing by the Vibrio histidine kinase VbrK

Abstract The ability of pathogens to develop resistance mechanisms makes the continuous search for novel therapeutic targets indispensable. Resistance-activating systems are promising targets for restoring the efficacy of existing antibiotics. VbrK/VbrR is a two-component system from Vibrio parahaemolyticus reported as the first sensing system in gram-negatives that directly detects β-lactam antibiotics. β-Lactam-induced activation of this system results in the expression of the serine β-lactamase CARB. In this study, we provide insights into the mechanism of β-lactam binding to the periplasmic sensor domain of the histidine kinase VbrK. Our results demonstrate that the interaction depends on the redox state of cysteines C86 and C107, highlighting the role of disulfide bond dynamics in modulating ligand recognition. We further show that formation of a non-covalent complex leads to acylation of the sensor domain by β-lactams, a modification that is slowly reversed through de-acylation, yielding the hydrolyzed β-lactam ring and allowing for recovery from induction once the antibiotic has been depleted from the environment. Together, these findings reveal a previously unrecognized redox- and covalent chemistry–dependent mode of β-lactam interaction with histidine kinases, providing a molecular framework to understand how VbrK detects and responds to β-lactam antibiotics, and opening new avenues to prevent manifestation of resistance. Short broader audience statement Bacterial pathogens can sense the presence of antibiotics and trigger resistance responses, making infections increasingly difficult to treat. In this study, we uncover a previously unknown mechanism by which β-lactam antibiotics interact with a sensor protein that activates resistance in Vibrio parahaemolyticus . These findings pave the way for designing new compounds that can block this interaction and thus restore the effectiveness of β-lactam antibiotics against gastrointestinal infections caused by resistant bacteria.