Quercetin Reduces the Virulence of S. aureus by Targeting ClpP to Protect Mice from MRSA-Induced Lethal Pneumonia

Abstract

The dramatic increase of methicillin-resistant Staphylococcus aureus (MRSA) poses a great challenge to the treatment of Staphylococcus aureus (S. aureus) infections. Therefore, there is an urgent need to identify novel anti-infective agents to attack new targets to overcome antibiotic resistance. Casein hydrolase P (ClpP) is a key virulence factor in S. aureus to maintain cellular homeostasis. We screened from flavonoids and finally determined that quercetin could effectively attenuate the virulence of MRSA. The results of the thermal shift assay showed that quercetin could bind to ClpP and reduce the thermal stability of ClpP, and the K_D value between quercetin and ClpP was 197 nM as determined by localized surface plasmon resonance. We found that quercetin exhibited a protective role of a mouse model of MRSA-induced lethal infection in a murine model. Based on the above facts, quercetin, as a ClpP inhibitor, could be further developed as a potential candidate for antivirulence agents to combat S. aureus infections. IMPORTANCE The resistance of Staphylococcus aureus (S. aureus) to various antibiotics has increased dramatically, and thus the development of new anti-infective drugs with new targets is urgently needed to combat resistance. Caseinolytic peptidase P (ClpP) is a casein hydrolase that has been shown to regulate a variety of important virulence factors in S. aureus. Here, we found that quercetin, a small-molecule compound from traditional Chinese herbal flavonoids, effectively inhibits ClpP activity. Quercetin attenuates the expression of multiple virulence factors in S. aureus and effectively protects mice from lethal pneumonia caused by MRSA. In conclusion, we determined that quercetin is a ClpP inhibitor and an effective lead compound for the development of a virulence factor-based treatment for S. aureus infection.

Keywords: MRSA; antivirulence; caseinolytic peptidase P; inhibitor; pneumonia.

FIG 1

Quercetin was found to be an inhibitor of ClpP from natural products. (a) Structure of quercetin. (b) The IC₅₀ value of quercetin on ClpP was (13.98 ± 1.25 μg/mL) based on the fluorescent substrate Suc-LY-AMC. (c) The growth of USA300 is unaffected by 64 μg/mL quercetin. Wild type USA300 was used as a control. (d) Quercetin at 64 μg/mL has no effect on the viability of Vero cells.

FIG 2

Quercetin significantly reduces the virulence of S. aureus in vitro. (a) Expression levels of agr, RNAIII, hla, luks, psm-α and spa were determined by qPCR in the presence of 32 μg/mL of quercetin. (b-e) Quantification of alpha-toxin and PVL expression levels in S. aureus USA300 and Newman under the effect of different concentrations of quercetin by Western blotting, and their corresponding gray value analysis. (f) Urease production in USA300 was induced by quercetin. ΔclpP served as a positive control. (g and h) The effect of different concentrations of quercetin on the hemolytic capacity of S. aureus USA300 and USA300 supernatant. (i) Effect of quercetin on the ability of S. aureus to invade A549 cells. A549 cells were lysed, and intracellular bacteria were determined by plate counting. (j) Flow cytometry determination of FITC-labeled SpA on the surface of S. aureus and their corresponding relative fluorescence intensity. ΔclpP served as a positive control. (k) Fluorescence microscope image of mouse macrophages J774 cells stained with live/dead assay. The green and red fluorescence indicated live and dead cells, respectively. (l) LDH release is presented as % of total LDH. Significance is calculated based on one-way ANOVA: **, P < 0.01 and ***, P < 0.001.

FIG 3

Quercetin can bind to ClpP. (a) The binding between quercetin and ClpP was examined by TSA. Quercetin caused a decrease in the thermal stability of ClpP. (b and c) SDS-PAGE analysis (b) and thermal shift assay curves (c) showing that quercetin caused a decrease in the T_m of ClpP protein in the cell lysates. The complete gel image is shown in Fig. S1. (d) Surface plasmon resonance imaging reveals the kinetics of quercetin binding to ClpP. The chromaticity lines represent the response of the dynamical signal. (e) T (Total) indicates the amount of protein initially added to ClpP and ClpX. W (Wash) indicates the effluent from the ClpXP complex after flowing through the Ni-NTA and eluting with a binding buffer without imidazole. E (Elute) indicates the effluent eluted with a binding buffer containing high concentration of imidazole. Quercetin could bind ClpP to dissociate ClpP from the ClpX complex. ADEP4 served as a positive control. Significance is calculated based on two-tailed t test: **, P < 0.01 and ***, P < 0.001.

FIG 4

Molecular modeling of ClpP-quercetin binding. (a-d) The results of the predicted molecular docking of M21, myricetin, alpinetin and quercetin with ClpP protein and the structural formulae of each compound, respectively. M21 and myricetin were used as positive controls. (e) The inhibition of ClpP activity by structurally similar flavonoids was determined by Suc-LY-AMC. Myricetin was used as positive control. (f) Two mutants Q47A-ClpP and S22A-ClpP were resistance to quercetin inhibition. Significance is calculated based on two-tailed t test: *, P < 0.05 and ***, P < 0.001.

FIG 5

Quercetin protects mice from MRSA pneumonia. (a) Survival of mice treated with quercetin (100 mg/kg) at the indicated times after infection with WT (USA300, 2e8 CFU/30 μL). Significance (P value) in the panels except (a) is calculated using log-rank test: **, P < 0.01 and ***, P < 0.001. (b) Gross and histopathology of S. aureus WT and WT-ΔclpP infected lung tissue from mice. Quercetin (100 mg/kg) treatment by subcutaneous injection. Scale bar, 50 μm. (c) The infectious bacterial load in the lung of mice with quercetin (100 mg/kg) treatment. In the graph, horizontal bars indicate the mean of bacterial load measurements, each dot represents a mouse. Significance is calculated based on one-way ANOVA: **, P < 0.01 and ***, P < 0.001.