Epigallocatechin gallate inhibits Streptococcus pneumoniae virulence by simultaneously targeting pneumolysin and sortase A

Abstract

Streptococcus pneumoniae (pneumococcus), the causative agent of several human diseases, possesses numerous virulence factors associated with pneumococcal infection and pathogenesis. Pneumolysin (PLY), an important virulence factor, is a member of the cholesterol-dependent cytolysin family and has cytolytic activity. Sortase A (SrtA), another crucial pneumococcal virulence determinate, contributes greatly to the anchoring of many virulence-associated surface proteins to the cell wall. In this study, epigallocatechin gallate (EGCG), a natural compound with little known antipneumococcal activity, was shown to directly inhibit PLY-mediated haemolysis and cytolysis by blocking the oligomerization of PLY and simultaneously reduce the peptidase activity of SrtA. The biofilm formation, production of neuraminidase A (NanA, the pneumococcal surface protein anchored by SrtA), and bacterial adhesion to human epithelial cells (Hep2) were inhibited effectively when S. pneumoniae D39 was cocultured with EGCG. The results from molecular dynamics simulations and mutational analysis confirmed the interaction of EGCG with PLY and SrtA, and EGCG binds to Glu277, Tyr358, and Arg359 in PLY and Thr169, Lys171, and Phe239 in SrtA. In vivo studies further demonstrated that EGCG protected mice against S. pneumoniae pneumonia. Our results imply that EGCG is an effective inhibitor of both PLY and SrtA and that an antivirulence strategy that directly targets PLY and SrtA using EGCG is a promising therapeutic option for S. pneumoniae pneumonia.

Keywords: Streptococcus pneumoniae; antivirulence; epigallocatechin gallate; neuraminidases A; pneumolysin; sortase A.

Figure 1

EGCG inhibits PLY haemolytic activity and SrtA_ΔN81 peptidase activity. (A) Chemical structure of EGCG. (B) Western blot analysis of PLY expression in culture precipitates of Streptococcus pneumoniae D39 treated with the indicated concentrations of EGCG. (C) Haemolysis assays were performed with purified PLY‐WT in the presence of various concentrations of EGCG using rabbit red blood cells in PBS. (D) The inhibition of SrtA_ΔN81 peptidase activity by EGCG. Purified SrtA_ΔN81‐WT and various concentrations of EGCG were incubated for 30 min. at 37°C, followed by the addition of SrtA_ΔN81 substrates and the fluorescent peptide Dabcyl‐QALPETGEE‐Edans and incubation for 1 hr. Finally, the fluorescence values of the reaction system were measured (excitation and emission wavelengths of 350 and 520 nm, respectively). The bars show the mean values of three independent assays. The error bars indicate the standard deviations (S.D.). * indicates P < 0.05 and ** indicates P < 0.01 compared with the drug‐free group, according to 2‐tailed Student's t‐tests. EGCG: epigallocatechin gallate, PLY: pneumolysin, SrtA: sortase A.

Figure 2

EGCG inhibits the oligomerization of PLY and the localization of NanA. (A) The inhibitory effect of EGCG on PLY oligomerization. Purified PLY‐WT was incubated with 4.36 and 8.73 μM EGCG, and the oligomerization of PLY was analysed by Western blot. (B) The influence of EGCG on NanA localization and SrtA expression in Streptococcus pneumoniae. D39 cells were cocultured with the indicated concentrations of EGCG and centrifuged, and the cell‐associated proteins were analysed by Western blot with murine anti‐NanA and anti‐SrtA serum. PLY was used as the loading control and detected with a monoclonal antibody. NanA: neuraminidase A. EGCG: epigallocatechin gallate.

Figure 3

The binding modes of PLY‐EGCG (A) and SrtA‐EGCG (B). The binding sites of EGCG with PLY (Ser256, Glu277, Tyr358 and Arg359) or SrtA (Thr167, Lys169 and Phe237) are identified. EGCG: epigallocatechin gallate.

Figure 4

Mutation of binding sites impairs the inhibitory effects of EGCG on PLY and SrtA_ΔN81. The inhibitory effects of various concentrations of EGCG on the haemolytic activities of purified PLY‐WT, PLY‐E277A, PLY‐Y358A and PLY‐R359A (A) were measured by haemolysis assays. The inhibitory effects of various concentrations of EGCG on the peptidase activities of purified SrtA_ΔN81‐WT, SrtA_ΔN81‐T169A, SrtA_ΔN81‐K171A and SrtA_ΔN81‐F239A (B) were measured by fluorescence resonance energy transfer (FRET) assays. The bars show the mean values of three independent assays. The error bars indicate the standard deviation (S.D.). * indicates P < 0.05 and ** indicates P < 0.01 compared with the drug‐free group according to 2‐tailed Student's t‐tests. EGCG: epigallocatechin gallate.

Figure 5

EGCG attenuates PLY‐mediated human alveolar epithelial (A549) cell injury. A549 cells were stained with a live (green)/dead (red) reagent 5 hrs after treatment with PLY, and images were captured using a confocal laser scanning microscope. (A) Untreated A549 cells. (B) Cells treated with 0.00078% (v/v) DMSO. (C) Cells treated with PLY‐WT in the absence of EGCG. D‐E, Cells treated with PLY‐WT pre‐incubated with 2.18 μΜ (D) and 8.73 μΜ (E) EGCG. The results shown in A–E are representative of the results from three independent experiments. LDH release by A549 cells treated with PLY‐WT (F), PLY‐E277A (G), PLY‐Y358A (H) or PLY‐R359A (I) pre‐incubated with the indicated concentrations of EGCG. The error bars indicate the S.D. * indicates P < 0.05 and ** indicates P < 0.01 compared with the drug‐free group according to 2‐tailed Student's t‐tests. EGCG: epigallocatechin gallate.

Figure 6

EGCG inhibits pneumococcal biofilm formation and adhesion to human epithelial cells. Streptococcus pneumoniae biofilms were grown in the presence of the indicated concentrations of EGCG for 18 hrs and detected by crystal violet staining and CFU counting. (A) Photograph of a biofilm stained with crystal violet. (B) Quantification of biofilm formation by measuring bound crystal violet. (C) Quantification of biofilm biomass by CFU counts. The biomass in the biofilm grown without EGCG was set as 100% for statistical analysis. (D) D39 cells pre‐cultured with the indicated concentrations of EGCG were added to Hep2 cells and then cocultured with the corresponding concentrations of EGCG for 2 hrs. After washing, D39 cells adherent to Hep2 cells were calculated by colony counting. The results shown in A are representative of the results from three independent experiments. The bars show the mean values of three independent assays. The error bars indicate the S.D. * indicates P < 0.05 and ** indicates P < 0.01 compared with the drug‐free group according to 2‐tailed Student's t‐tests. PC: Positive Control; NC: Negative Control. EGCG: epigallocatechin gallate.

Figure 7

EGCG protects mice against Streptococcus pneumoniae pneumonia. BALB/c mice were inoculated with S. pneumoniae via the intranasal route and treated subcutaneously with PBS or EGCG. Each group contained 10 mice. At 48 hrs post‐inoculation, the mice were killed, the gross pathological changes (A) and histopathology (B) of lung tissue were assessed, and the bacterial burden (D) and wet/dry weight ratio (E) of lungs were calculated. Lung tissues were stained with haematoxylin and eosin (original magnification, ×100). (C) The mortality of mice infected with pneumococci was supervised for 120 hrs. The results shown in (A) and (B) are representative of the results from three independent experiments. The bars in (D and E) show the mean values of three independent assays. The error bars indicate the S.D. * indicates P < 0.05 and ** indicates P < 0.01 compared with the drug‐free group according to 2‐tailed Student's t‐tests. EGCG: epigallocatechin gallate.