<i>gltB</i> encoding glutamate synthase is involved in persister and biofilm formation and virulence in <i>Staphylococcus aureus</i>

Glutamate metabolism plays a pivotal role in linking the tricarboxylic acid cycle, arginine biosynthesis, and purine metabolism, and these pathways have been shown to be involved in persister formation. However, the relationship among glutamate metabolism, bacterial antibiotic tolerance, and virulence remains unclear. In this study, <i>gltB,</i> which encodes the large subunit of glutamate synthase in <i>Staphylococcus aureus,</i> was knocked out. The ∆<i>gltB</i> mutant in the stationary phase showed less tolerance to antibiotics and was killed completely after exposure to lethal doses of ampicillin and norfloxacin after 11 and 6 days, respectively, while the parent strain still had abundant viable bacteria. The <i>gltB</i> complemented strain restored antibiotic tolerance. Interestingly, exogenous glutamate supplementation of ∆<i>gltB</i> restored the tolerance to antibiotics. Moreover, ∆<i>gltB</i> is more susceptible to heat, carbon starvation, and oxidative stress. Furthermore, the ability of ∆<i>gltB</i> to coagulate plasma, produce staphyloxanthin, and form biofilms was significantly weakened. In addition, ∆<i>gltB</i> attenuated virulence in BALB/c mice, and its 50% lethal dose (LD50) (1.14 × 10¹⁰ CFU/mL, 95% CI: 7.29 × 10⁹-2.75 × 10¹⁰) was higher than that of the parent strain (2.39 × 10⁹ CFU/mL, 95% CI: 9.99 × 10⁸-4.42 × 10⁹). The expression levels of major virulence genes, including <i>eta, hla, hlgA, hlgC, lukD, lukE, lukS, lukF,</i> and <i>sea,</i> as well as staphyloxanthin synthesis-related genes, including <i>crtM</i> and c<i>rtQ,</i> were significantly downregulated in ∆<i>gltB</i>. This study revealed that <i>gltB</i> is involved in both antibiotic tolerance and virulence in <i>S. aureus</i> and provides new insights into the mechanism of persister formation and virulence, with implications for the development of novel drugs.IMPORTANCE<i>Staphylococcus aureus</i> is a leading bacterial cause of death, and persister formation renders it tolerant to antibiotics and is associated with its persistent infections. Glutamate metabolism plays a critical role in linking the tricarboxylic acid cycle, arginine biosynthesis, and purine metabolism, and these pathways have been shown to be involved in persister formation. This work first discovered that <i>gltB</i>, the large subunit of glutamate synthase gene in <i>S. aureus</i>, is involved in tolerance to antibiotics and heat, carbon starvation, and oxidative stress. Furthermore, the <i>gltB</i> mutant attenuated virulence in mice, owing to the inhibition of glutamate synthesis, which significantly weakened the ability of <i>S. aureus</i> to coagulate plasma, produce staphyloxanthin, form biofilms, and express virulence factors. These findings confirm the important role of glutamate metabolism in the formation of persister and virulence in <i>S. aureus</i> and provide new targets for developing novel anti-persister and anti-virulence drugs.