Myricetin Acts as an Inhibitor of Type II NADH Dehydrogenase from Staphylococcus aureus

Abstract

Background: Staphylococcus aureus is a common pathogenic microorganism in humans and animals. Type II NADH oxidoreductase (NDH-2) is the only NADH:quinone oxidoreductase present in this organism and represents a promising target for the development of anti-staphylococcal drugs. Recently, myricetin, a natural flavonoid from vegetables and fruits, was found to be a potential inhibitor of NDH-2 of S. aureus. The objective of this study was to evaluate the inhibitory properties of myricetin against NDH-2 and its impact on the growth and expression of virulence factors in S. aureus.

Results: A screening method was established to identify effective inhibitors of NDH-2, based on heterologously expressed S. aureus NDH-2. Myricetin was found to be an effective inhibitor of NDH-2 with a half maximal inhibitory concentration (IC₅₀) of 2 μM. In silico predictions and enzyme inhibition kinetics further characterized myricetin as a competitive inhibitor of NDH-2 with respect to the substrate menadione (MK). The minimum inhibitory concentrations (MICs) of myricetin against S. aureus strains ranged from 64 to 128 μg/mL. Time-kill assays showed that myricetin was a bactericidal agent against S. aureus. In line with being a competitive inhibitor of the NDH-2 substrate MK, the anti-staphylococcal activity of myricetin was antagonized by MK-4. In addition, myricetin was found to inhibit the gene expression of enterotoxin SeA and reduce the hemolytic activity induced by S. aureus culture on rabbit erythrocytes in a dose-dependent manner.

Conclusions: Myricetin was newly discovered to be a competitive inhibitor of S. aureus NDH-2 in relation to the substrate MK. This discovery offers a fresh perspective on the anti-staphylococcal activity of myricetin.

Keywords: Staphylococcus aureus; Type II NADH dehydrogenase; anti-virulence activity; competitive inhibitor; myricetin.

Figure 1

Structure of myricetin.

Figure 2

Preparation and enzymatic properties of heterologously expressed NDH−2. (A) SDS−PAGE of purified protein. 1: Lysate, 2: Supernatant, 3: Pellet, 4: Wash, 5: 30 mM imidazole, 6: 80 mM imidazole, 7: 150 mM imidazole. (B) Western blotting of NDH−2 using anti−6His−tag antibody. a: 80 mM imidazole, b: 150 mM imidazole, M: Protein Marker. (C,D) The plot of the reaction rate (V) vs. substrate concentration. (E,F) The kinetic parameters Km were calculated by Lineweaver−Burk plot of 1/V vs. 1/S. (G) The turnover number (kcat) and affinity (Km) for the substrates of the enzyme were determined. Data are expressed as mean ± SD of three independent experiments.

Figure 3

The effect of myricetin on enzyme activity of NDH−2. Concentration of NADH was set at 300 μM for all experiments. A variable slope model was fitted to determine the IC₅₀ values. In each panel, NADH: quinone oxidoreduction activities in the absence of drugs were used for 100%. (A) The NADH UV absorbance at 340 nm was monitored after the addtion of various agents; (B) The NADH UV absorbance at 340 nm was monitored after the addtion of myricetin; (C) The NADH UV absorbance at 340 nm was monitored after the addtion of HQNO; (D) Myricetin inhibition curves for NDH−2 in the presence of 30 μM MK; (E) HQNO inhibition curves for NDH−2 in the presence of 30 μM MK.

Figure 4

Myricetin acting as a competitive inhibitor of NDH−2 substrate MK. (A) Electronic cloud plot of NDH−2 and the interaction of myricetin with NDH−2 (PDB code 5AN1). (B) Michaelis−Menten plot of myricetin against NDH−2. (C) Lineweaver−Burk plot of myricetin against NDH−2.

Figure 5

The antimicrobial activity of myricetin against Staphylococcus aureus. (A,B) Time–kill curves for Staphylococcus aureus treated with myricetin. The concentrations are as follows: ●, 3.2% DMSO; ▴, 2 × MIC of myricetin; ▾, 4 × MIC of myricetin; ■, 2 × MIC of vancomycin. (C,D) MK4 antagonizes the antibacterial activity of myricetin.

Figure 6

Effect of myricetin on Staphylococcus aureus toxins. Changes in the transcript levels of crtM (A), hlα (B), seA (E), and seB (F) in myricetin-treated S. aureus were analyzed relative to those in untreated control cultures. Relative normalized expression values were determined by RT-qPCR. (C) Bacterial growth was measured by optical density at 600 nm. (D) Hemolytic activity of myricetin-treated Staphylococcus aureus. Data represent means ± SD (n = 3). Statistically significant differences between differences in drug concentrations are shown as * p < 0.1, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.