Taxifolin, an Inhibitor of Sortase A, Interferes With the Adhesion of Methicillin-Resistant Staphylococcal aureus

Abstract

The evolution and spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant hidden risk to human public health. The majority of antibiotics used clinically have become mostly ineffective, and so the development of novel anti-infection strategies is urgently required. Since Staphylococcus aureus (S. aureus) cysteine transpeptidase sortase A (SrtA) mediates the surface-anchoring of proteins to its surface, compounds that inhibit SrtA are considered potential antivirulence treatments. Herein, we report on the efficacy of the potent SrtA inhibitor taxifolin (Tax), a flavonoid compound isolated from Chinese herbs. It was able to reversibly block the activity of SrtA with an IC₅₀ of 24.53 ± 0.42 μM. Tax did not display toxicity toward mammalian cells or S. aureus at a concentration of 200 μM. In addition, Tax attenuated the virulence-related phenotype of SrtA in vitro by decreasing the adherence of S. aureus, reducing the formation of a biofilm, and anchoring of S. aureus protein A on its cell wall. The mechanism of the SrtA-Tax interaction was determined using a localized surface plasmon resonance assay. Subsequent mechanistic studies confirmed that Asp-170 and Gln-172 were the principal sites on SrtA with which it binds to Tax. Importantly, in vivo experiments demonstrated that Tax protects mice against pneumonia induced by lethal doses of MRSA, significantly improving their survival rate and reducing the number of viable S. aureus in the lung tissue. The present study indicates that Tax is a useful pioneer compound for the development of novel agents against S. aureus infections.

Keywords: antivirulence; inhibitor; methicillin-resistant Staphylococcus aureus; pneumonia; sortase A; taxifolin.

FIGURE 1

Inhibition of SrtA transpeptidation. (A) Chemical structure of Tax. (B) Tax inhibits SrtA cleavage of a fluorogenic peptide substrate (Abz-LPATG-Dnp-NH₂) in a dose-dependent manner in vitro. Each reaction condition was assayed in triplicate from which the IC₅₀ was determined. (C) Recombinant SrtA was treated with 10 × IC₅₀ of Tax and then diluted, after which transpeptidation activity was measured using a FRET assay. Untreated SrtA (Control) represented 100% activity.

FIGURE 2

Growth curve and cytotoxicity. (A) Growth curves of S. aureus USA300 and the ΔsrtA group with different concentrations of Tax. (B) Growth rate of S. aureus USA300 and S. aureus treated with Tax (25 μM). (C) Percent cell viability of HEK293 (purple) and HepG2 (blue) cells were measured using a CCK-8 assay after incubation with Tax for 24 h.

FIGURE 3

Effect of SrtA inhibitor on virulence-related phenotypes in S. aureus Newman strain. (A) Impact of Tax on the ability of S. aureus to adhere to fibrinogen. (B) Biofilm formation of S. aureus in the presence of different concentrations of Tax. (C) Mature biofilms of S. aureus in the presence of different concentrations of Tax. The ΔsrtA group represented the positive control. (D) Tax affects internalization of S. aureus into A549 cells. A549 cells were infected with S. aureus pretreated with different concentrations of Tax then lysed 2 h post-infection. The number of viable S. aureus in the cells was quantified by serial dilution on TSA agar plates. (E) Effects of Tax on S. aureus protein A (SpA) using FITC-labeled rabbit IgG. Error bars represent means ± SD of three replicates. *P < 0.05, **P < 0.01, ***P < 0.001 calculated with a two-tailed Student’s t-test.

FIGURE 4

Expression of SrtA in the presence of Tax and the interaction of different concentrations of Tax with SrtA. (A) Western blot analysis of SrtA protein from S. aureus treated with various concentrations of Tax and grayscale analysis of SrtA protein bands. (B) LSPR analysis verified the binding affinity of Tax with SrtA. Error bars represent means ± SD of three replicates. ***P < 0.001 calculated with a two-tailed Student’s t-test.

FIGURE 5

Molecular modeling of the interaction between Tax and SrtA. (A) RMSF (Å) graph of free-SrtA (black) and the SrtA-Tax (red) complex during 40-ns MD. (B) Docking model of Tax with SrtA during a molecular dynamics simulation. (C) Binding free energy decomposition in each residue between Tax and modeled S. aureus SrtA.

FIGURE 6

Effect of treatment with Tax on S. aureus-induced pneumonia in mice. (A) Effect of Tax on the survival of mice (n = 10 per group) infected with a lethal dose of S. aureus. (B) Effect of Tax (100 mg/kg) on bacterial load in the lungs of mice (n = 6). (C) Gross pathological changes and histopathology of mouse lungs treated with Tax (100 mg/kg/d) or untreated. Scale bar: 50 μm. (D) Lung wet-to-dry weight ratio (W/D). Each value represents mean ± SD (n = 5 per group). **P < 0.01, ***P < 0.001; Mann-Whitney test, two-tailed. Horizontal bars represent mean values. Animal data were obtained from two separate experiments.

FIGURE 7

Experimental flow chart.