Punicalagin, an Inhibitor of Sortase A, Is a Promising Therapeutic Drug to Combat Methicillin-Resistant Staphylococcus aureus Infections

Abstract

Antimicrobial resistance (AMR) poses a major threat to human health globally. Staphylococcus aureus is recognized as a cause of disease worldwide, especially methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). The enzyme sortase A (SrtA), present on the cell surface of S. aureus, plays a key role in bacterial virulence without affecting the bacterial viability, and SrtA-deficient S. aureus strains do not affect the growth of bacteria. Here, we found that punicalagin, a natural compound, was able to inhibit SrtA activity with a very low half maximal inhibitory concentration (IC₅₀) value of 4.23 μg/mL, and punicalagin is a reversible inhibitor of SrtA. Moreover, punicalagin has no distinct cytotoxicity toward A549, HEK293T, or HepG2 cells at a much higher concentration than the IC₅₀ detected by MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assays. In addition, punicalagin visibly attenuated the virulence-related phenotype of SrtA in vitro by decreasing adhesion of S. aureus to fibrinogen, reducing the ability of protein A (SpA) displayed on the surface of the bacteria and biofilm formation. Fluorescence quenching elucidated the interaction between punicalagin and SrtA. Molecular docking further implied that the inhibitory activity lay in the bond between punicalagin and SrtA residues LYS190, TYR187, ALA104, and GLU106. In In vivo studies, we surprisingly found that punicalagin had a more effective curative effect combined with cefotaxime when mice were infected with pneumonia caused by MRSA. Essentially, punicalagin, a therapeutic compound targeting SrtA, demonstrates great potential for combating MRSA infections.

Keywords: Staphylococcus aureus; antivirulence; cefotaxime; methicillin resistance; punicalagin; sortase A.

FIG 1

Punicalagin inhibits the activity of SrtA. (A) Screening of SrtA and AgrA inhibitors from hundreds of natural compounds by FRET and CETSA. When inhibition activity was greater than 60%, the compound was considered a potential SrtA inhibitor. (B) The structure chart of punicalagin with its PubChem CID and molecular weight and the High Performance Liquid Chromatography (HPLC) of punicalagin is provided in Fig. S1. (C) A FRET assay was used to determine that punicalagin is an inhibitor of SrtA with an IC₅₀ of 4.23 μg/mL. (D) The growth curve of S. aureus USA300 with or without punicalagin (32 μg/mL). (E to G) Effects of punicalagin on the cytotoxicity of A549 (E), HEK293T (F), and HepG2 cells (G) determined by MTT assays.

FIG 2

The effect of punicalagin on SrtA-related phenotypes of S. aureus. (A) Effect of punicalagin on the adhesion of S. aureus USA300 to fibrinogen. (B) Effect of punicalagin on the internalization of S. aureus into A549 cells. (C) Effect of punicalagin on S. aureus USA300 biofilm formation. (D) Effect of S. aureus surface protein (SpA) stained with FITC-labeled rabbit IgG detected by flow cytometry analysis. (E) Live/dead reagent-A549 cells were observed with fluorescent imaging. (F) The LDH release by A549 cells treated with punicalagin. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the untreated group. The experiment was repeated at least three times.

FIG 3

Punicalagin combined to SrtA of S. aureus USA300. (A) Western blot assay to detect the expression of StrA in the S. aureus USA300 with different concentrations of punicalagin (0 to 32 μg/mL) (B) Binding affinity between punicalagin and SrtA determined by fluorescence quenching. The K_A was calculated by plotting the Stern-Volmer SrtA quenching. (C) Reversible inhibitory effect of punicalagin on SrtA. (D) The 3D structural determination of a SrtA with punicalagin complex by a molecular modeling method. (E) The inhibitory activity of WT SrtA and SrtA mutants (A104G-srtA, E106A-srtA, Y187A-srtA, and K109A-srtA) determined by FRET. ***, P < 0.001, calculated using one-way analysis of variance (ANOVA). (F to I) Binding affinity between punicalagin and SrtA mutants determined by fluorescence quenching. (J and K) The 3D structural determination of a SrtA with berberine chloride (J) or thiadiazole complex (K) by a molecular docking method.

FIG 4

Punicalagin protected mice from S. aureus infection. (A) Schematic diagram of a survival and mouse pneumonia model. (B) Effect of punicalagin on survival after 96 h in C57BL/6J mice (n = 10). **, P < 0.01; ***, P < 0.001 compared with the USA300 group. (C) The bacterial load of lung tissue (n = 5). (D) The histopathology of lung tissue detected by HE staining (n = 5). Scale bars, 1 cm and 100 μm. (E to G) ELISA for the secretion level of inflammatory factors (IFN-γ, IL-6, and TNF-α) in the alveolar lavage fluid of each group of mice (n = 3).

FIG 5

Punicalagin reduces the formation of biofilm of S. aureus and attenuates the adhesion and invasion to the host and the anchoring of surface proteins by inhibiting SrtA.