Mechanistic origins and evolutionary erosion of collateral sensitivity in a β-lactamase

As antibiotic discovery stalls, exploiting collateral sensitivity, where resistance to one drug increases sensitivity to another, offers a promising route to extend the lifespan of existing drugs. However, the molecular origins and robustness of such trade-offs at the level of single resistance determinants remain poorly understood. Here, we examined a previously evolved trajectory of the {beta}-lactamase OXA-48 to Q4 (A33V/F72L/T212A/S213A), which confers a 40-fold increase in ceftazidime resistance when expressed in Escherichia coli. This evolution coincided with the emergence of collateral sensitivity to piperacillin (27-fold reduction), likely caused by the introduction of F72L. We challenged the stability of this collateral sensitivity network by subjecting Q4 to directed evolution under co-selective pressure from both ceftazidime and piperacillin. This selected for the substitution V120G, which alleviated the piperacillin trade-off while maintaining elevated resistance to ceftazidime in genetic backgrounds harboring F72L. Structural and computational analyses revealed that, during evolution from OXA-48 to Q4, F72L introduced substantial conformational changes in the {Omega}-loop, likely leading to less productive piperacillin binding poses. V120G counteracted the effect of F72L by decreasing the {Omega}-loops conformational freedom, thereby partially restoring piperacillin resistance. Finally, we show that other substitutions at position 120 can exert similar mitigating effects. Taken together, our results provide a mechanistic understanding of how adaptive solutions both generate and erode collateral sensitivity, knowledge crucial for predicting the long-term stability of these networks. IMPORTANCEThe evolution of antimicrobial resistance to one drug can also increase bacterial susceptibility to other drugs, a phenomenon known as collateral sensitivity. Understanding the mechanisms underlying these effects may help clinicians design antimicrobial treatment strategies that limit the emergence of resistance. However, for clinical applications, collateral effects must be stable and robust over a certain evolutionary time. While many studies have focused on the stability of collateral effects at the bacterial population level, their stability during the evolution of a single resistance determinant has remained largely unknown. Here, we studied the molecular origins of collateral sensitivity arising during the evolution of the {beta}-lactamase OXA-48 and robustness of this network under co-selection. By linking resistance evolution to changes in protein structure, we illuminate the role of active site loop dynamics in shaping both the collateral effect and its mitigation. Such results are important for understanding collateral sensitivity-based strategies and how they may shape the evolutionary trajectories of resistance enzymes.