Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress

Reviewer #1 (Public Review):

As a pathogen, S. aureus has evolved strategies to evade the host's immune system. It effectively remains 'under the radar' in the host until it reaches high population densities, at which point it triggers virulence mechanisms, enabling it to spread within the host. The agr quorum sensing system is central to this process, as it coordinates the pathogen's virulence network in response to its cell density. When a threshold cell density (quorum) is reached, individual cells in the population detect the quorum peptide (AgrD). This activates the Agr two-component system comprising of a histidine kinase (AgrC) and response regulator (AgrA), leading to the expression of exoenzymes and toxins that facilitate the pathogenicity of S. aureus.
However, previous research has indicated that oxidative stress can possibly bypass agr quorum signaling and inhibit agr-dependent gene expression. Specifically, when exposed to high concentrations of hydrogen peroxide (H2O2), the redox-sensitive cysteines in AgrA can form an intramolecular disulfide bond, preventing AgrA from binding to DNA and initiating gene expression. Moreover, another study has shown that cells which have responded to the quorum peptide are vulnerable to oxidative damage. This damage is mediated by PSM toxins that are produced by quorum peptide responders. Consequently, this oxidative stress leads to the selection of agr mutants that are better adapted to growth in oxygen-rich environments and can exploit the benefits of the products released by the quorum-responding cells.

Given this sensitivity of the agr system to oxidative modification and the fitness cost arising from PSM expression, it raises a pertinent question: how do cells with a functional agr quorum sensing system persist within the population under conditions of oxidative stress without being overtaken by agr mutants? The current study by Podkowik et al. offers a plausible explanation. It suggests that cells that respond to the quorum peptide may be primed against oxidative stress by activating intrinsic mechanisms that reduce not only the endogenous production of harmful ROS but also mitigate their adverse effects on the cell, thus providing a unique benefit to cells that maintain an active agr system. Interestingly, these protective mechanisms are long-lived, and safeguard the cells against external oxidative stressors such as H2O2, even after the agr system has been deactivated in the population.

In their study, the authors present compelling evidence that supports the role of agr in shielding S. aureus from lethal H2O2 stress, and they establish that this protection is connected to the activation of agr-dependent RNAIII and the subsequent block of Rot translation. Importantly, the protective mechanisms that are activated persist throughout growth and provide S. aureus with defense against the host's ROS in a murine intraperitoneal infection model.

However, the study falls short in elucidating the specific intrinsic mechanisms responsible for this long-lasting protection against external ROS. While the authors infer that agr mutants, which are more vulnerable to external H2O2, display an increased respiratory activity and gene expression profile associated with aerobic fermentation, it remains ambiguous whether controlling these mechanisms alone can confer extended protection from external H2O2 in an aerobic environment. Further research is needed to confirm this hypothesis.

In summary, this study reveals that the agr quorum sensing system's role in relation to ROS is multifaceted and more complex than previously thought, as it orchestrates a balance between mechanisms that both induce and mitigate endogenous ROS, ultimately contributing to the pathogenesis of S. aureus.

Reviewer #2 (Public Review):

In their study, Podkowik et al. elucidate the protective role of the accessory gene regulator (agr) system in Staphylococcus aureus against hydrogen peroxide (H2O2) stress. Their findings demonstrate that agr safeguards the bacterium by controlling the accumulation of reactive oxygen species (ROS), independent of agr activation kinetics. This protection is facilitated through a regulatory interaction between RNAIII and Rot, impacting virulence factor production and metabolism, thereby influencing ROS levels. Notably, the study highlights the remarkable adaptive capabilities of S. aureus conferred by agr. The protective effects of agr extend beyond the peak of agr transcription at high cell density, persisting even during the early log-phase. This indicates the significance of agr-mediated protection throughout the infection process. The absence of agr has profound consequences, as observed by the upregulation of respiration and fermentation genes, leading to increased ROS generation and subsequent cellular demise. Interestingly, the study also reveals divergent effects of agr deficiency on susceptibility to hydrogen peroxide compared to ciprofloxacin. While agr deficiency heightens vulnerability to H2O2, it also upregulates the expression of bsaA, countering the endogenous ROS induced by ciprofloxacin. These findings underscore the complex and context-dependent nature of agr-mediated protection. Furthermore, in vivo investigations using murine models provide valuable insights into the importance of agr in promoting S. aureus fitness, particularly in the context of neutrophil-mediated clearance, with notable emphasis on the pulmonary milieu. Overall, this study significantly advances our understanding of agr-mediated protection in S. aureus and sheds light on the sophisticated adaptive mechanisms employed by the bacterium to fortify itself against oxidative stress encountered during infection.

The conclusions of this paper are mostly well supported by the data; however, certain aspects regarding the impact of agr loss on bacterial metabolic status require additional experimental clarification.

1. The RNA-seq analysis revealed that the Δagr strain exhibited increased expression of genes involved in respiration and fermentation, suggesting enhanced energy generation. However, metabolic modeling based on transcriptomic data indicated a decrease in tricarboxylic acid (TCA) cycle and lactate flux per unit of glucose uptake in the Δagr mutant. Additionally, intracellular ATP levels were significantly lower in the Δagr mutant compared to the wild-type strain, despite the carbon being directed into an acetate-generating, ATP-yielding carbon "overflow" pathway. Furthermore, growth analysis in nutrient-constrained medium demonstrated a decrease in the growth rate and yield of the Δagr mutant. Given that S. aureus actively utilizes the electron transport chain (ETC) to replenish NAD pools during aerobic growth on glucose, supporting glycolytic flux and pyruvate dehydrogenase complex (PDHC) activity while restricting TCA cycle activity through carbon catabolite repression (CCR), it is suggested that the authors analyze glucose consumption rates in conjunction with the determination of intracellular levels of pyruvate, AcCoA, and TCA cycle intermediates such as citrate and fumarate. These additional experiments will provide valuable insights into the metabolic fate of glucose and pyruvate and their subsequent impact on cellular respiration and fermentation in the Δagr mutant.

2. The authors highlighted the importance of redox balance in Δagr cells by emphasizing the tendency of these cells to prioritize NAD+-generating lactate production over generating additional ATP from acetate. However, the results regarding acetate and lactate production in Δagr cells during aerobic growth suggest that carbon is directed towards acetate generation rather than lactate.

3. The authors mentioned that respiration and fermentation typically reduce the NAD+/NADH ratios, and since these activities are elevated in Δagr strains (Figure 5F-G), they initially anticipated a lower NAD+/NADH ratio compared to wild-type agr cells. However, the increase in respiration and activation of fermentative pathways leads to a decrease in NADH levels, therefore resulting in an increase in the NAD+/NADH ratio.