Molecular Characterization of Clinical Rel Mutations and Consequences for Resistance Expression and Fitness in Staphylococcus aureus

The stringent response (SR) is a universal stress response that acts as a global regulator of bacterial physiology and virulence, and is a contributor to antibiotic tolerance and resistance. In most bacteria, the SR is controlled by a bifunctional enzyme, Rel, which both synthesizes and hydrolyzes the alarmone (p)ppGpp via two distinct catalytic domains. The balance between these antagonistic activities is fine-tuned to the needs of the cell and, in a "relaxed" state, the hydrolase activity of Rel dominates. We have previously shown that two single amino acid substitutions in Rel (that were identified in clinical isolates from persistent infections) confer elevated basal concentrations of (p)ppGpp and consequent multidrug tolerance in Staphylococcus aureus. Here, we explore the molecular details of how these mutations bring about this increase in cellular (p)ppGpp and investigate the wider cellular consequences in terms of resistance expression, resistance development, and bacterial fitness. Using enzyme assays, we show that both these mutations drastically reduce the hydrolase activity of Rel, thereby shifting the balance of Rel activity in favor of (p)ppGpp synthesis. We also demonstrate that these mutations induce high-level, homogeneous expression of beta-lactam resistance and confer a significant fitness advantage in the presence of bactericidal antibiotics (but a fitness cost in the absence of antibiotic). In contrast, these mutations do not appear to accelerate the emergence of endogenous resistance mutations in vitro. Overall, our findings reveal the complex nature of Rel regulation and the multifaceted implications of clinical Rel mutations in terms of antibiotic efficacy and bacteria survival.