Hinokiflavone Attenuates the Virulence of Methicillin-Resistant Staphylococcus aureus by Targeting Caseinolytic Protease P

Abstract

Drug-resistant bacteria was the third leading cause of death worldwide in 2019, which sounds like a cautionary note for global public health. Therefore, developing novel strategies to combat Methicillin-resistant Staphylococcus aureus (MRSA) infections is the need of the hour. Caseinolytic protease P (ClpP) represents pivotal microbial degradation machinery in MRSA involved in bacterial homeostasis and pathogenicity, considered an ideal target for combating S. aureus infections. Herein, we identified a natural compound, hinokiflavone, that inhibited the activity of ClpP of MRSA strain USA300 with an IC₅₀ of 34.36 μg/mL. Further assays showed that hinokiflavone reduced the virulence of S. aureus by inhibiting multiple virulence factors expression. Results obtained from cellular thermal transfer assay (CETSA), thermal shift assay (TSA), local surface plasmon resonance (LSPR) and molecular docking (MD) assay enunciated that hinokiflavone directly bonded to ClpP with confirmed docking sites, including SER-22, LYS-26 and ARG-28. In vivo, the evaluation of anti-infective activity showed that hinokiflavone in combination with vancomycin effectively protected mice from MRSA-induced fatal pneumonia, which was more potent than vancomycin alone. As mentioned above, hinokiflavone, as an inhibitor of ClpP, could be further developed into a promising adjuvant against S. aureus infections.

Keywords: Caseinolytic protease P; hinokiflavone; infection; methicillin-resistant Staphylococcus aureus; pneumonia.

FIG 1

Hinokiflavone was identified as an inhibitor of S. aureus ClpP. (a) Diagram of FRET assay for screening ClpP inhibitors. When inhibition activity was greater than 60%, the compound was considered a potential ClpP inhibitor. (b) Chemical structure of hinokiflavone. (c) Hinokiflavone inhibited the cleavage of the fluorescent substrate Suc-LY-AMC by ClpP and the IC₅₀ of hinokiflavone was 34.36 μg/mL. (d) The growth of USA300 was not affected by 64 μg/mL of hinokiflavone. Wild-type USA300 was supplemented with 0.5% DMSO as a control group. (e) Effect of different concentrations of hinokiflavone on the viability of HEK-293T cells.

FIG 2

Hinokiflavone depressed the expression of various virulence factors regulated by ClpP in S. aureus. (a) Transcript levels of the related genes agr, RNAIII, hla, luks, psm-α, spa, and clpP were determined by RT-qPCR exposed to the concentration of hinokiflavone at 64 μg/mL. The reference gene used in Quantitative Real-time PCR is 16sRNA. (b) Quantification of the effect of different concentrations of hinokiflavone on the expression of alpha-toxin and (c) PVL in S. aureus USA300 by Western blotting and statistical analysis of their corresponding gray-scale values. (d) Quantification of the effect of different concentrations of hinokiflavone on the expression of alpha-toxin and (e) PVL in S. aureus Newman by Western blotting and statistical analysis of their corresponding gray-scale values. (f) The effect of hinokiflavone on the urease production in USA300 and ΔclpP strains. (g) Effect of different concentrations of hinokiflavone on the hemolysis activity of S. aureus USA300. (h) Determination of the neutralizing activity of hinokiflavone on Hla in supernatant of S. aureus USA300. Data were expressed as mean ± SD for three independently experiments. *, P < 0.05, **, P < 0.01 and ***, P < 0.001 compared to the control group.

FIG 3

Hinokiflavone binded to ClpP and inhibited its activity. (a) The binding of different concentrations of hinokiflavone to ClpP was detected by TSA technique. Hinokiflavone inhibited the thermal stability of ClpP in a concentration-dependent manner. (b) Grayscale images by SDS-PAGE and the quantification of CETSA of ClpP with and without hinokiflavone (128 μg/mL) incubation. (c) Molecular docking simulates the key amino acid sites for the binding of hinokiflavone and ClpP. (d) Three potential binding sites mutants of K26Α-ClpP, R28Α-ClpP and S46Α-ClpP showed increased resistance to ClpP inhibition by hinokiflavone. (e) LSPR assay confirmed that a direct interaction between hinokiflavone and ClpP. (f) The main mechanism of hinokiflavone inhibiting S. aureus ClpP and the biological consequence. Significance of data are expressed as mean ± SD. *, P < 0.05, **, P < 0.01 and ***, P < 0.001.

FIG 4

Synergistic protective effect of hinokiflavone and vancomycin against pneumonia induced by S. aureus in mice. (a) Experimental model of pneumonia induced by MRSA in C57BL/6J mice. (b) Effect of hinokiflavone treatment (100 mg/kg·d⁻¹), vancomycin (100 mg/kg·d⁻¹) or hinokiflavone combination with vancomycin treatment on the survival of C57BL/6J mice (n = 10) exposed to lethal doses of S. aureus USA300. (c) After 48 h of treatment, lung tissue from each group of mice (n = 5) was homogenized and bacterial load quantified. (d) Gross and histopathological examination of lung tissue from S. aureus WT and WT-ΔclpP infected mice treated with hinokiflavone (100 mg/kg·d⁻¹), vancomycin (100 mg/kg·d⁻¹) or a combination of hinokiflavone and vancomycin by subcutaneous injection. Scale bar, 100 μm. (e-g) Levels of IFN-γ, IL-6 and TNF-α, inflammatory cytokines, in lung perfusion fluid of mice in each group (n = 3). Data are means ± SD of results for compared with USA300 + PBS, *, P < 0.05, **, P < 0.01 and ***, P < 0.001 (by one-way ANOVA); compared with USA300 + Vancomycin, #, P < 0.05 (by unpaired t test). Each experiment was independently duplicated three times.