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

Abstract

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H₂O₂, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr increased both respiration and fermentation but decreased ATP levels and growth, suggesting that Δagr cells assume a hyperactive metabolic state in response to reduced metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H₂O₂ doses. Increased survival of wild-type agr cells during H₂O₂ exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H₂O₂. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived "memory" of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Nox2^-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Keywords: Staphylococcus aureus; agr; peroxide (H2O2); quorum-sensing; reactive oxygen species (ROS).

Figure 1.. agr protects from killing by H₂O₂ throughout the growth cycle.

(A) Effect of culture growth phase. Overnight cultures of S. aureus LAC wild-type (WT, BS819) or Δagr (BS1348) were diluted (OD₆₀₀~0.05) into fresh TSB medium and grown with shaking from early exponential (1 h, OD₆₀₀~0.15) through late log (5 h, OD₆₀₀~4) phase. At the indicated times, early (undiluted) and late exponential phase cultures (diluted into fresh TSB medium to OD₆₀₀~0.15) were treated with H₂O₂ (20 mM). After 60 min, aliquots were removed, serially diluted, and plated for determination of viable counts. Percent survival was calculated relative to a sample taken at the time of H₂O₂ addition. (B) Kinetics of killing by H₂O₂. Wild-type and Δagr mutant strains were grown to early exponential (OD₆₀₀~0.15) and treated with 20 mM H₂O₂ for the times indicated, and percent survival was determined by plating. (C) Effect of H₂O₂ concentration on survival. Cultures prepared as in panel B were treated with the indicated peroxide concentrations for 60 min prior to plating and determination of percent survival. (D) Complementation of agr deletion mutation. Cultures of wild-type (WT) cells (BS819), Δagr mutant (BS1348), and complemented Δagr mutant carrying a chromosomally integrated wild-type operon (pJC1111-agrI) were treated with 20 mM H₂O₂ for 60 min followed by plating to determine percent survival. Data represent the means ± S.D. from biological replicates (n = 3).

Figure 2.. Involvement of RNAIII and rot-dependent pathways in agr-mediated protection from H₂O₂-mediated killing.

Cultures were grown for 1 h following dilution from overnight cultures to early log phase (OD₆₀₀~0.15) and then treated with 20 mM H₂O₂ for 60 min before determination of percent survival by plating and enumeration of colonies. (A) Wild-type LAC (WT, BS819), Δagr mutant (BS1348), and ΔrnaIII mutant (GAW183). (B) Δrot and Δagr Δrot double mutant (BS1302). (C) Wild-type (WT) strain Newman (NM, BS12), Δagr mutant (BS13), and ΔRNAIII mutant (BS669). (D) Overexpression of rot. Rot was expressed from a plasmid-borne wild-type rot (pOS1-Plgt-rot, strain VJT14.28). Data represent the means ± S.D. from biological replicates (n = 3).

Figure 3.. agr-mediated protection from H₂O₂ stress is uncoupled from agr activation kinetics.

(A) Assay design. An ΔagrBD deletion mutant (GAW130) was complemented in trans by the autoinducing product (AIP) of AgrBD in an ΔrnaIII (GAW183) mutant that produces AIP endogenously; AgrC activation in the ΔagrBD strain leads to downstream activation of RNAIII. The agrBD strain, engineered in-frame to avoid polar effects on downstream genes agrC and agrA, senses but does not produce autoinducer. The ΔrnaIII mutant, constructed by replacement of rnaIII with a cadmium resistance cassette (rnaIII::cadA), produces autoinducer but lacks RNAIII, the effector molecule of agr-mediated phenotypes with respect to H₂O₂. The image was created using BioRender (BioRender.com). (B) Trans-activation demonstrated by hemolysin activity on sheep blood agar plates. Bottom of figure shows zone of clearing (hemolysin activity) after mixing 10⁸ ΔagrBD CFU with an equal number of ΔrnaIII. Zone of clearance is a consequence of AgrC receptor activation in trans by AIP produced by the ΔrnaIII mutant. (C) Absence of trans-activation with short-term culture. The wild-type strain RN6734 (WT, BS435), ΔrnaIII (GAW183), ΔagrBD (GAW130), and ΔrnaIII and ΔagrBD mutants were mixed 1:1 immediately before growth from overnight culture. Overnight cultures were diluted (OD₆₀₀~0.05) into fresh TSB medium, mixed, and grown to early log phase (OD₆₀₀~0.15) when they were treated with 20 mM H₂O₂ for 60 min and assayed for percent survival by plating. (D) Kinetics of killing by H₂O_2. Survival assays employing ΔrnaIII and ΔagrBD mixtures, performed as in panel C, but grown from early exponential (1 h, OD₆₀₀~0.15) through late log (5 h, OD₆₀₀~4) phase in TSB. Cultures were treated with H₂O₂ (20 mM for 1 h) at the indicated time points. (E) Proportion of mixed population for panel D represented by each mutant after incubation. The ΔagrBD mutant contained an erythromycin-resistance marker to distinguish the strains following plating of serial dilutions on TS agar with or without erythromycin (5 μg/ml). Data represent the means ± S.D. from biological replicates (n = 3). (F) Trans-activation during long-term culture. The wild-type strain RN6734 (WT, BS435), ΔrnaIII (strain GAW183), ΔagrBD (strain GAW130), and ΔrnaIII and ΔagrBD mutants mixed 1:1 prior to overnight culture. Survival assays employing ΔrnaIII and ΔagrBD mixtures, performed as in panel C. (G) Kinetics of killing by H₂O₂. Survival assays employing ΔrnaIII and ΔagrBD mixtures, performed as in panel D. Cultures were treated with H₂O₂ (20 mM for 1 h) at the indicated time points. (H) Proportion of mixed population for panel G represented by each mutant after incubation, performed as in panel E. Data represent the means ± S.D. from biological replicates (n = 3).

Figure 4.. Association of agr deficiency with increased expression of respiration and fermentation genes during aerobic growth.

(A) Relative expression of respiration and fermentation genes. RNA-seq comparison of S. aureus LAC wild-type (WT, BS819) and Δagr mutant (BS1348) grown to late exponential phase (OD₆₀₀~4.0). Shown are significantly up-regulated genes in the Δagr mutant (normalized expression values are at least twofold higher than in the wild-type). Heatmap colors indicate expression z-scores. RNA-seq data are from three independent cultures. See Supplementary file 1 for supporting information. (B) Schematic representation of agr-induced changes in metabolic flux, inferred from transcriptomic data (Supplementary file 1) by SPOT (Simplified Pearson correlation with Transcriptomic data). Metabolic intermediates and enzymes involved in catalyzing reactions are shown. The magnitude of the flux (units per 100 units of glucose uptake flux) is denoted by arrowhead thickness. Boxed charts indicate relative flux activity levels in wild-type versus Δagr strains. Enzyme names are linked to abbreviations in boxed charts (e.g., lactate dehydrogenase, LDH). See Supplementary file 2 for supporting information. (C) RNA-seq comparison of an Δagr Δrot double mutant (BS1302) with its parental Δagr strain (BS1348). Heatmap colors indicate expression z-scores. Sample preparation and figure labelling as for Figure 4A. See Supplementary file 3 for supporting information.

Fig. 5.. Association of agr deficiency with a metabolic flux shift toward fermentive metabolism during aerobic growth.

(A) Intracellular ATP levels. Comparison of S. aureus LAC wild-type (WT, BS819) and Δagr mutant (BS1348) strains for ATP expressed as μg/10⁸ cells after growth of cultures in TSB medium to late-exponential phase (OD₆₀₀~4.0). (B) Extracellular acetate levels. Samples were taken after 1, 2, 3, 4, and 24 h of growth in TSB medium; strains were wild-type (WT, BS819) and Δagr mutant (BS1348). (C-D) Extracellular lactate and acetate levels during low oxygen culture. S. aureus LAC wild-type (WT, BS819) and Δagr mutant (BS1348) were grown in TSB medium with suboptimal aeration to late-exponential phase (4 h, OD₆₀₀~4.0). (E-F) Oxygen consumption. Strains LAC wild-type (WT, BS819) and Δagr mutant (BS1348) were compared using Seahorse XFp analyzer (F), and the rate of oxygen consumption (E) was determined from the linear portion of the consumption curve. Representative experiments from at least 3 independent assays are shown. (G-H) NAD⁺ and NADH levels. Colorimetric assay of NAD⁺ (G) and NADH levels (H) for S. aureus wild-type (WT, BS819) and Δagr mutant (BS1348) after growth of cultures to late-exponential phase (OD₆₀₀~4.0). (I) NAD⁺/NADH ratio. For all panels, data points are the mean value ± SD (n = 3). *P < 0.05; ****P < 0.0001, by Student’s two-tailed t test. Seahorse statistical significances are compared to TSB medium.

Figure 6.. Increase in ROS levels associated with Δagr deficiency.

Flow cytometry measurements. S. aureus LAC wild-type (WT, BS819) and Δagr mutant (BS1348) were grown overnight, diluted, cultured in TSB medium for 1 h, and treated with carboxy-H2DCFDA (10 μM) for 5 min. Relative cell number is on the vertical axis. Unst. indicates samples containing LAC wild-type cells not treated with carboxy-H2DCFDA. (B) Five replicate experiments gave similar results (“fold change” indicates the mean wild-type or Δagr ROS level divided by the mean autofluorescence background signal; lines connect results in replicate experiments).

Figure 7.. Rot-mediated up-regulation of H₂O₂-stimulated genes relative to those in an agr mutant.

Genes shown are those up-regulated in a Δagr Δrot double mutant (BS1302) relative to that observed with the Δagr strain (BS1348). H₂O₂ treatment was for 30 min. Peroxide concentrations for Δagr (2.5 mM H₂O₂) and Δagr Δrot (10 mM H₂O₂) were determined to achieve ~50% cell survival [see Methods and Figure 7—figure supplement 1]). RNA-seq data are from three independent cultures. Heatmap colors indicate expression z-scores. See Supplementary file 3 for supporting information.

Figure 8.. Involvement of endogenous ROS in agr-mediated protection from lethal H₂O₂ stress.

(A) Protective effect of menadione on survival. S. aureus LAC wild type (BS819) and Δagr mutant (BS1348) cultures were grown to late exponential phase (4 h after dilution of overnight cultures), exposed to 80 μM menadione (MD) with or without 4 mM N-acetyl cysteine (NAC) for 30 min prior to treatment with H₂O₂ (20 mM for 1 h) and measurement of survival. (B) Effect of sodA deletion on survival. Cultures of wild-type (BS819), Δagr (BS1348), a sodA::tetM (BS1422), and sodA::tetM-agr double mutant (BS1423) were grown to early (1 h after dilution, OD₆₀₀~0.15) or late log (4 h after dilution, OD₆₀₀~4.0) prior to treatment with 20 mM H₂O₂ for 60 min. (C) Effect of H₂O₂ concentration on survival. Late log (4 h, OD₆₀₀~4.0) cultures of the wild-type and Δagr mutant strains were treated with indicated concentrations of H₂O₂ for 60 min. (D) Complementation of sodA deletion mutation. A plasmid-borne wild-type sodA gene was expressed under control of the sarA constitutive promoter (pJC1111-sodA) in late log-phase (4 h, OD₆₀₀~4.0) cells treated with 20 mM H₂O₂ for 60 min. (E) SodA activity. Wild-type or the indicated mutants were grown to late-exponential phase (OD₆₀₀~4.0); Sod activity was measured as in Methods. (F) Effect of ahpC deletion on survival. Late log-phase cultures of wild-type (BS819), Δagr (BS1348), ahpC::bursa (BS1486), and ΔahpC::bursa-agr double-mutant (BS1487) cells were treated with 20 mM H₂O₂ for 60 min. (G) Effect of ahpC deletion on expression of katA in the indicated mutants. Total cellular RNA was extracted from late exponential-phase cultures (OD₆₀₀~4.0), followed by reverse transcription and PCR amplification of the indicated genes, using rpoB as an internal standard. mRNA levels were normalized to those of each gene to wild-type control. Data represent the means ± S.D. from (n = 3) biological replicates. One-way ANOVA was used to determine statistical differences between samples (****P < 0.0001).

Figure 9.. Survival advantage of agr priming of S. aureus absent in phagocyte NADPH-deficient murine infection.

(A) percentage of ΔagrBD (AIP-responsive in-frame deletion mutant carrying an intact RNAIII) cells and (B) bacterial burden in lung or spleen after 2 h of intraperitoneal infection of wild-type (WT) C57BL/6 mice or phagocyte NADPH oxidase-deficient (Cybb^−/−) mice (see Figure 9—figure supplement 1 for data with other organs). ΔagrBD and ΔrnaIII mutant cultures were grown separately and mixed at a 1:1 ratio either before (primed) or after (unprimed) overnight growth, as for Figure 3. Both primed and unprimed mixtures were diluted after overnight growth, grown to early log phase (OD₆₀₀~0.15), and used as inocula (1 × 10⁸ CFU) for intraperitoneal infection (n = 2 groups of 10 mice each). After 2 h, lungs and spleen were harvested and homogenized; aliquots were diluted and plated to enumerate viable bacteria. Output ratios and total and mutant CFU from tissue homogenates were determined as for Fig 4E and 4H. A Mann-Whitney test (panel 9A) or Student’s two-tailed t test (panel 9B) were used to determine the statistical significance of the difference between primed and unprimed cultures. Error bars indicate standard deviation (**P < 0.01; ****P < 0.0001).

Figure 10.. Schematic representation of agr-mediated protection from ROS.

At low levels of oxidative stress, the redox sensor in AgrA binds to DNA at promoters P2 and P3, activating expression of the two operons. Expression of RNAIII blocks translation of Rot, which decreases respiration and production of superoxide. ROS quenchers (sodA and katA/ahpC) suppress formation of most ROS that would otherwise signal the redox sensor in AgrA to halt stimulation of RNAIII expression and the production of further superoxide via respiration. This feedback system regulates respiration thereby limiting the accumulation of ROS in wild-type cells. Wild-type cells are primed for induction of protective genes (e.g., clpB/C, dps) by loss of the rot repressor system via an unknown mechanism when cells experience damage from high levels of oxidative stress (experimentally introduced as lethal exogenous H₂O₂); Δagr cells that experience high levels of endogenous H₂O₂ fail to induce protective genes. Exogenous H₂O₂ or high levels of endogenous ROS, for example from extreme stress due to ciprofloxacin (12), lower RNAIII expression and allow Rot to stimulate bsaA expression, which produces a protective antioxidant. The protective action of an ahpC deficiency acts through compensatory expression of katA, which results in more effective scavenging of H₂O₂ produced from increased respiration in Δagr strains and/or exogenous lethal H₂O₂.

Figure 11.. Relationship of agr priming and virulence.

The ecology of abscess formation and subsequent bacterial dissemination can be described as a cycle. (a) During abscess formation, a hallmark of S. aureus disease, agr is activated by high bacterial cell density (quorum sensing) (63). (b) The bacterium assumes a primed stage due to repression of the rot repressor. (c, d, e) The lethal effects of immune challenge, which is called triggering (64), are survived by the persistence (“memory”) of the agr-activated state. (f) agr expression is inactivated by oxidation, thereby elevating expression of the antioxidant bsaA (7), which enables proliferation when oxidative stress is sublethal (7). (g) By surviving damage caused by lethal exogenous oxidative stress, primed S. aureus escape from the localized abscess to produce new infectious lesions (bloodstream dissemination) or to infect new hosts, where the cycle would be repeated.