Biochemical characterization and inhibition of thermolabile hemolysin from Vibrio parahaemolyticus by phenolic compounds

Abstract

Vibrio parahaemolyticus (Vp), a typical microorganism inhabiting marine ecosystems, uses pathogenic virulence molecules such as hemolysins to cause bacterial infections of both human and marine animals. The thermolabile hemolysin VpTLH lyses human erythrocytes by a phospholipase B/A2 enzymatic activity in egg-yolk lecithin. However, few studies have been characterized the biochemical properties and the use of VpTLH as a molecular target for natural compounds as an alternative to control Vp infection. Here, we evaluated the biochemical and inhibition parameters of the recombinant VpTLH using enzymatic and hemolytic assays and determined the molecular interactions by in silico docking analysis. The highest enzymatic activity was at pH 8 and 50 °C, and it was inactivated by 20 min at 60 °C with Tm = 50.9 °C. Additionally, the flavonoids quercetin, epigallocatechin gallate, and morin inhibited the VpTLH activity with IC50 values of 4.5 µM, 6.3 µM, and 9.9 µM, respectively; while phenolics acids were not effective inhibitors for this enzyme. Boltzmann and Arrhenius equation analysis indicate that VpTLH is a thermolabile enzyme. The inhibition of both enzymatic and hemolytic activities by flavonoids agrees with molecular docking, suggesting that flavonoids could interact with the active site's amino acids. Future research is necessary to evaluate the antibacterial activity of flavonoids against Vp in vivo.

Keywords: Inhibition; Molecular docking; Phenolic compounds; SGHN phospholipases; Thermal stability; Thermolabile-hemolysin.

Figure 1. Recombinant overexpression, purification, and refolding of VpTLH.

(A) 12% SDS-PAGE of the recombinant over-expression process and in vitro refolding of purified VpTLH. M, molecular weight marker; lane 1 and 2, the soluble and insoluble fractions of non-induced E. coli culture. Lane 3 and 4, insoluble and soluble fraction 16 hours after the addition of IPTG to the culture; lane 5, solubilized inclusion bodies (8M urea); lane 6, purified VpTLH under denaturing conditions; lane 7, in vitro refolded VpTLH. (B) Chromatogram of VpTLH IMAC purification under denaturing conditions. (C) Esterase activity assay of refolded Vp TLH. 410 nm absorbance increases as PNPL hydrolysis releasing p-nitrophenol. The assay was performed by triplicate; PNPL self-hydrolysis (control) was assayed without Vp TLH. (D) Effect of lecithin on VpTLH enzymatic activity.

Figure 2. Biochemical properties of Vp TLH.

Enzymatic activity was calculated as the residual activity respect to the highest value detected in each assay. Results were the mean ± SE (n = 3). (A) pH effect on enzymatic activity, a different buffer, was a function of pH evaluated as described in the ‘Materials and Methods’ section. (B) Activity profile at different temperatures. Cell holder temperature within the reaction cell was stabilized by 60-sec min each assay. (C) The plot of linearized Arrhenius equation, a temperature in which enzymatic activity starts decreasing (inflection point), was fitted to a linear model (R² = 0.985). lnK, the natural logarithm of initial velocities; temperatures were in Kelvin degrees. (D) Thermal stability of VpTLH, data were fitted to the Boltzmann sigmoidal model (R² = 0.99). All data were analyzed in Prism5 Graphpad^® program.

Figure 3. Effect of substrate concentration (PNPL) on Vp TLH enzymatic activity.

Fitting data calculated Michaelis-Menten kinetics parameters to non-linear regression model (R² = 0.9851). All substrate concentrations were assayed by triplicate.

Figure 4. Effect of phenolic acids on Vp TLH enzymatic activity.

Vp TLH activity was assayed in the presence of each phenolic acid and the final concentration as indicated. Residual activity was calculated based on Vp TLH activity under optimal assay conditions in the absence of phenolics acids. Results are mean SE (n = 3) statistical differences (p < 0.05) compared to control without phenolics acids as denoted with an asterisk. Control (-), VpTLH without phenolic acids; GA, gallic acid; PR, protocatechuic acid; CL, chlorogenic acid and VA, vanillic acid.

Figure 5. Dose-response analysis of Vp TLH inhibition by flavonoids.

Vp TLH enzymatic activity was assayed (n = 3) in presence of each flavonoid; data were fitted (R² > 0.95) to the dose-response model to calculate IC50 values. Residual activity was calculated as a percentage considering VpTLH enzymatic activity in the absence of tested flavonoids as 100%. EGCG, Epigallocatechingallate.

Figure 6. Inhibition of Vp TLH hemolytic activity by flavonoids.

Each inhibitor concentration was assayed in triplicate. Bars represented SEM. Hemolytic activity in the presence of flavonoids was calculated as percentage respect to VpTLH without inhibitors (CN). Inhibitor concentrations with statically significant differences (p < 0.05) compared to control are denoted with an asterisk.

Figure 7. Predicted Structure of the Vp TLH.

(A) Overall structure superposition of the VpTLH and the VvTLH. N- and C-terminal domains are showed by magenta/cyan (Vp) and gray/orange (Vv). The cylinders colored by atom type shows the catalytic triad (Ser-His-Asp). (B) Superposition of the catalytic amino acids VpTLH (carbon atoms in gray) and Vv (carbon atoms are purple). (C) Hydrogen bonds (continuous lines) interactions in catalytic amino acids of VpTLH. (D) Charge surface representationVp TLH catalytic site cavity (indicated by the arrow).

Figure 8. Molecular docking (A) and interaction maps (B) of substrates into Vp TLH active site.

PC, phosphatidylcholine and PNPL, p-nitrophenylaurate. The protein molecule is displayed as a surface in white and ligand as a cylinder colored by atom type with carbon atoms in green. Interaction maps were showed depicted by color as follows: hydrogen bonds (green), alkyl (pink), saline bridge (orange), and Van der Waals interactions (light green).

Figure 9. Molecular docking (A) and interaction map (B) of flavonoids into the Vp TLH active site.

EGCG = epigallocatechin gallate. The protein molecule is displayed as a surface in white and ligand as a cylinder colored by atom type with carbon atoms in green. Interaction maps were depicted by color as follows: hydrogen bonds (green), π-alkyl (pink), π-anion (orange), unfavorable donor/acceptor hydrogen bond (red), and Van der Waals interaction (light green).