## 3 Discussion
First, _relQ_ transcription is known to be responsive to specific environmental stress signals and may be governed by promoter elements less susceptible to repression by membrane or metabolic perturbations beyond a threshold level. Second, feedback regulation through (p)ppGpp levels or sigma factor activity could stabilize _relQ_ expression once an initial suppression occurs. Notably, [PERSON] et al. [14] reported that _relQ_ is inducible under specific conditions of amino acid starvation and oxidative stress, which may buffer it against further downregulation in the presence of sustained metabolic inhibition. Lastly, functional overlap between _relP_ and _relQ_ might allow the cell to maintain a basal (p)ppGpp synthesis capability by preserving minimal _relQ_ expression, particularly in the face of external modulators such as diosgenin. These hypotheses underscore the complexity of stringent response regulation and warrant further transcriptional profiling studies under varying stress and inhibitor conditions. Additionally, the differential response of _relP_ and _relQ_ to diosgenin treatment suggests that distinct regulatory mechanisms govern the expression of these synthases, as was suggested in a previous study [14]. Another major finding in this study was that diosgenin treatment significantly reduces intracellular ATP levels in _S. aureus_ (Figure 4). Treatment with 80 uM diosgenin reduced intracellular ATP levels by 36%, while 160 uM diosgenin decreased ATP levels by 38%. These reductions suggest that diosgenin disrupts ATP synthesis pathways, potentially limiting the energy resources required for persister cell formation and maintenance. The similar extent of ATP reduction at both diosgenin concentrations indicates that diosgenin'sinhibitory effect may reach a plateau at or before 80 uM. It should be noted that the results in this study were calculated based on the ATP concentration in the lysate solution obtained after cell disruption, not the intracellular ATP concentration. To estimate ATP concentration on a per-cell basis, the measured ATP concentration was divided by the cell concentration. Given the critical role of ATP in supporting the dormancy and resuscitation of persister cells, the observed depletion may significantly impact persister cell survival. Moreover, when coupled with the decreased expression of _relP_ and _relQ_, these findings suggest that diosgenin disrupts the metabolic framework that underpins stringent response signaling. Previous studies have proposed that disrupting the membrane integrity of persister cells can sensitize them to antibiotics by enhancing intracellular drug uptake [27]. Diosgenin did not significantly alter membrane permeability at either 80 or 160 uM (Figure 5A), indicating that its anti-persister activity is not mediated through membrane disruption. However, diosgenin markedly decreased membrane fluidity in a dose-dependent manner, with the 80 uM treatment reducing fluidity by 35% and the 160 uM treatment reducing it by 41% (Figure 5B). The extent of membrane rigidification at 160 uM was comparable, though in the opposite direction, to the increase in membrane fluidity induced by benzyl alcohol, a known membrane fluidizer [29]. These reductions in membrane fluidity suggest that diosgenin interacts with membrane lipids, potentially altering membrane-associated signaling pathways involved in persister cell regulation. It is interesting that, despite these changes, no significant alterations in membrane potential were detected at any concentration (Supplementary Figure S1). This finding indicates that under the tested conditions, diosgenin affects membrane dynamics but does not disrupt ion gradients or membrane polarization.