Galangin as a direct inhibitor of vWbp protects mice from Staphylococcus aureus-induced pneumonia

Abstract

The surge in multidrug resistance in Staphylococcus aureus (S. aureus) and the lag in antibiotic discovery necessitate the development of new anti-infective strategies to reduce S. aureus infections. In S. aureus, von Willebrand factor-binding protein (vWbp) is not only the main coagulase that triggers host prothrombin activation and formation of fibrin cables but also bridges the bacterial cell wall and von Willebrand factor, thereby allowing S. aureus to bind to platelets and endothelial cells, playing a vital role in pathogenesis of S. aureus infections. Here, we have identified that galangin, a bioactive compound found in honey and Alpinia officinarum Hance, is a potent and direct inhibitor of vWbp by coagulation activity inhibition assay, thermal shift assay and biolayer interferometry assay. Molecular dynamic simulations and verification experiments revealed that the Trp-64 and Leu-69 residues are necessary for the binding of galangin to vWbp. Significantly, galangin attenuated S. aureus virulence in a mouse S. aureus-induced pneumonia model. In addition, we also identified that galangin can enhance the therapeutic effect of latamoxef on S. aureus-induced pneumonia. Taken together, the results suggest that galangin may be used for the development of therapeutic drugs or utilized as adjuvants to combine with antibiotics to combat S. aureus-related infections.

Keywords: Staphylococcus aureus; galangin; pneumonia; von Willebrand factor-binding protein.

FIGURE 1

Galangin inhibits the activity of vWbp. (A) Structure of galangin. (B) Effects of various concentrations of galangin (0, 16, 32, 64, 128 and 256 μg/ml) on the coagulation activity of recombinant vWbp. (C) a, Clotting activity of different concentrations of vWbp (from well 5 to 1 are 10, 5, 2.5, 1.25 and 0.625 mg/mL, respectively.); c, 5 mg/ml vWbp was mixed with different concentrations of galangin (0, 16, 32, 64 and 128 μg/ml) and added to wells 1–5. Recording of the size of the coagulation zone (b, d). ^** p < 0.01 and ^*** p < 0.001 were calculated using one‐way ANOVO

FIGURE 2

Galangin does not inhibit bacterial growth and the expression of vWbp. (A) Growth kinetics of S. aureus Newman treated with or without galangin (256 μg/ml). S. aureus ΔvWbp served as a control. Data represent in mean values ±SD. (B) Expression of vWbp was detected by Western blot. S. aureus ΔvWbp served as a control. Sortase A (SrtA) was used as the loading control in the experiment. The results are representative of two independent assays. (C) Grey value analysis of vWbp protein. Error bars indicate mean ±SD of three independent experiments

FIGURE 3

Determination of the binding of galangin to vWbp. (A) Galangin enhanced the thermal stability of the vWbp protein. The thermal shift assay of vWbp was performed in the presence (red curve) or absence (black curve) of galangin. (B) Biolayer interferometry assay showed that the association and dissociation sensorgrams for the interaction of different concentrations of galangin to vWbp. The Ni‐NTA biosensor was exposed to five different concentrations of galangin, and the fitting of the data to the curve was performed with Forte'Bio analysis software

FIGURE 4

Binding mode of galangin with vWbp and verification of key residues involved in the interaction of inhibitor with vWbp. (A) RMSF of the residues of vWbp in the free protein and the vWbp‐galangin complex. (B) The binding energy decomposition of residues in the vWbp‐galangin complex. (C) The putative binding pattern of galangin and vWbp was obtained from an MD simulation. The clotting activity of WT vWbp and its mutant proteins in the absence (D) or presence (E) of galangin

FIGURE 5

Galangin has a protective effect on S. aureus infection in mice. (A) Mice were infected with S. aureus Newman or S. aureus Newman ΔvWbp via the intra‐nasal route. Survival rates of galangin‐treated or untreated mice. WT + DMSO versus WT + galangin, **p < 0.01, log‐rank test. (B) The numbers of bacteria in the lungs of infected mice 24 h of infection. ^***p < 0.001 versus DMSO‐treated mice (n = 10). p Values were calculated using one‐way ANOVO. (C) Gross pathological changes and histopathology of lung tissues in mice in different infected or treated groups. Scale bar, 100 μm. Animal data were obtained from two independent experiments

FIGURE 6

Galangin enhances the therapeutic effect of latamoxef on S. aureus infection. (A) Survival curves of mice that treated with or without galangin and/or latamoxef. WT + DMSO versus WT + combination, **p < 0.01; WT + galangin versus WT + combination, *p < 0.05; WT + latamoxef versus WT + combination, *p < 0.05, log‐rank test. (B) Bacteria burden in lungs of infected mice 24 h post‐infection. ^*** p < 0.001 versus the DMSO group (n = 10). (C) Gross pathological changes and histopathology of lung tissues treated with either galangin and/or latamoxef. Scale bar, 100 μm. Animal data were obtained from two independent experiments