Exploring the potential of isorhapontigenin: attenuating Staphylococcus aureus virulence through MgrA-mediated regulation

Abstract

The emerging prevalence of drug-resistant Staphylococcus aureus isolates underscores the urgent need for alternative therapeutic strategies due to the declining effectiveness of traditional antibiotics in clinical settings. MgrA, a key virulence regulator in S. aureus, orchestrates the expression of numerous virulence factors. Here, we report the discovery of isorhapontigenin, a methoxylated analog of resveratrol, as a potential anti-virulence agent against S. aureus. Isorhapontigenin effectively inhibits the hemolytic activity of S. aureus in a non-bactericidal manner. Additionally, it significantly reduces the cytotoxicity of S. aureus and impairs its ability to survive in macrophages. Mechanistically, isorhapontigenin modulates the expression of virulence factors, dose-dependently downregulating hla and upregulating the MgrA-regulated gene spa. Electrophoretic mobility shift assays demonstrated that isorhapontigenin inhibits the binding of MgrA to the hla promoter in a dose-dependent manner. Thermal shift assays confirmed the direct interaction between isorhapontigenin and the MgrA protein. The in vivo experiments demonstrated that isorhapontigenin significantly reduced the area of skin abscesses and improved survival in a pneumonia model while decreasing bacterial burden and inflammation in the lungs. In conclusion, isorhapontigenin holds potential as a candidate drug for further development as an anti-virulence agent for treating S. aureus infections.

Importance: The emergence of antibiotic-resistant Staphylococcus aureus strains presents a formidable challenge to public health, necessitating novel approaches in combating these pathogens. Traditional antibiotics are becoming increasingly ineffective, leading to a pressing need for innovative therapeutic strategies. In this study, targeting virulence factors that play a crucial role in the pathogenesis of bacterial infections offers a promising alternative to circumvent resistance mechanisms. The discovery of isorhapontigenin as an inhibitor of S. aureus virulence represents a significant advance in anti-virulence therapy.

Keywords: MgrA; Staphylococcus aureus; isorhapontigenin; virulence.

Fig 1

In vitro and in vivo toxicity of isorhapontigenin (ISO). (A) The chemical structures of isorhapontigenin. (B) Hemolytic activity of isorhapontigenin on rabbit erythrocytes. Positive control: 2% Triton X-100; negative control: PBS. (C) Cell viability of BEAS-2B and L-02 cell lines after 24-h exposure to various concentrations of isorhapontigenin, relative to untreated control cells (set at 100% viability). (D) Survival rates of G. mellonella larvae after administration of isorhapontigenin, with 10 individuals per treatment group. The positive control group received injections of S. aureus Newman, and the negative control group received PBS injections.

Fig 2

Effect of isorhapontigenin (ISO) on the hemolytic activity of S. aureus. (A) The effects of isorhapontigenin on the hemolytic activity of S. aureus Newman. Dilute the supernatant from bacterial cultures, both isorhapontigenin-treated and untreated, by a specified factor ×-fold dilution, and incubate with a 5% RBC suspension. Positive control: 2% Triton X-100; negative control: PBS. (B) Growth curves of S. aureus Newman cultured in the presence of 50 µM isorhapontigenin. DMSO serves as a control to exclude the effects of the solvent on bacterial growth, with TSB as a negative control. (C) RT-qPCR analysis of the effect of isorhapontigenin on the expression of the hla gene. (D) Western blot analysis of the impact of isorhapontigenin on the production of α-hemolysin. (E) Evaluation of isorhapontigenin’s effect on the hemolytic activity of clinical S. aureus isolates. Positive control: 2% Triton X-100; negative control: PBS.

Fig 3

Effect of isorhapontigenin (ISO) on the cytotoxicity and immune evasion capabilities of S. aureus. (A) The effects of isorhapontigenin on LDH release from BEAS-2B cells after infection with S. aureus for 24 h. LDH release is expressed as a percentage of the maximal release induced by untreated S. aureus Newman. (B) Effect of isorhapontigenin on S. aureus survival in whole blood. Survival rates are presented as percentages relative to the maximal survival of untreated S. aureus Newman. (C) Treatment with isorhapontigenin decreases S. aureus survival in RAW264.7 cells.

Fig 4

Isorhapontigenin (ISO) attenuating S. aureus virulence through MgrA-mediated regulation. (A) RT-qPCR analysis of the effect of isorhapontigenin on key virulence regulators and their target genes. (B) EMSA analysis of the impact of isorhapontigenin on MgrA protein binding to hla promoter. Lane 1 shows EMSA with protein only; lane 2 shows EMSA with hla DNA only; lanes 3–6 show EMSA with increasing concentrations of isorhapontigenin (0, 1, 25, and 50 µM). (C) TSA analysis of the interaction between isorhapontigenin and MgrA protein. (D) Molecular docking analysis of MgrA protein with isorhapontigenin.

Fig 5

Therapeutic effects of isorhapontigenin (ISO) against S. aureus Newman-induced skin abscess and pneumonia in mice. (A) Skin lesions resulting from S. aureus infection (photographed on day 3 post-infection). (B) Comparison of abscess size (area) between Newman (control) and isorhapontigenin-treated groups (treated with 20 mg/kg per day). (C) The impact of isorhapontigenin on survival rates of mice (n = 10) infected with a lethal dose of S. aureus Newman. (D) Gross evaluation of lungs in all mice and determination of bacterial load in lung tissue. (E) Histological analysis of the impact of isorhapontigenin on the lungs of mice, using H&E staining.