Activation of the Spx redox sensor counters cysteine-driven Fe(II) depletion under disulfide stress

In many low G+C Gram-positive bacteria, the global regulator Spx helps maintain thiol homeostasis during disulfide stress, when protein thiols form aberrant disulfide bonds that can lead to misfolding and oxidative damage. Spx-dependent gene expression is triggered when an intramolecular disulfide bond forms between two cysteines in its redox switch. Surprisingly, some Spx functions persist even in the absence of an active redox switch, highlighting the need to better understand the physiological significance of maintaining this regulatory feature. Here, we utilize a spxC10A mutant that encodes a redox-insensitive Spx variant to study the role of the Spx redox switch in Staphylococcus aureus. We show that the spxC10A mutant is hypersensitive to diamide-induced disulfide stress and exhibits widespread transcriptional dysregulation of genes that contribute to thiol maintenance and disulfide repair. Remarkably, the spxC10A mutant rapidly adapts to disulfide stress by increasing its intracellular pool of L-cysteine (L-Cys) through enhanced uptake, which helps restore a reduced intracellular environment. However, during this process increased L-Cys inadvertently depletes cytosolic Fe(II), leading to growth inhibition of the spxC10A mutant. Finally, we show that the Spx-dependent control of intracellular L-Cys is critical for S. aureus survival when it encounters human neutrophils. Overall, these findings suggest that staphylococcal adaptation to disulfide stress through intracellular L-Cys accumulation imposes significant fitness costs that S. aureus overcomes by rapid regulatory control of thiol homeostasis through a functional Spx redox switch. SignificanceAll cells have a pool of low molecular weight thiols, such as cysteine, glutathione, bacillithiol, and coenzyme A, to maintain redox balance under oxidative and disulfide stress. Among these, cysteine is a very effective thiol but is highly reactive, and its intracellular concentration must be tightly regulated. In S. aureus, we found that cysteine accumulates intracellularly during disulfide stress and if left unchecked, can inadvertently deplete cytosolic Fe(II), leading to growth inhibition. To prevent cysteine toxicity, S. aureus activates the global regulator Spx, which rapidly induces genes that restore thiol homeostasis and limits cysteine accumulation.