Novel Copper Oxide Bio-Nanocrystals to Target Outer Membrane Lectin of Vancomycin-Resistant Enterococcus faecium (VREfm): In Silico, Bioavailability, Antimicrobial, and Anticancer Potential

Abstract

In present study, we used Olea europaea leaf extract to biosynthesize in situ Copper Oxide nanocrystals (CuO @OVLe NCs) with powerful antibacterial and anti-cancer capabilities. Physio-chemical analyses, such as UV/Vis, FTIR, XRD, EDX, SEM, and TEM, were applied to characterize CuO @OVLe NCs. The UV/Vis spectrum demonstrated a strong peak at 345 nm. Furthermore, FTIR, XRD, and EDX validated the coating operation's contact with colloidal CuO @OVLe NCs. According to TEM and SEM analyses, CuO @OVLe NCs exhibited a spherical shape and uniform distribution of size with aggregation, for an average size of ~75 nm. The nanoparticles demonstrated a considerable antibacterial effect against E. faecium bacterial growth, as well as an increased inhibition rate in a dose-dependent manner on the MCF-7, PC3, and HpeG2 cancer cell lines and a decreased inhibition rate on WRL-68. Molecular docking and MD simulation were used to demonstrate the high binding affinity of a ligand (Oleuropein) toward the lectin receptor complex of the outer membrane to vancomycin-resistant E. faecium (VREfm) via amino acids (Leu 195, Thr 288, His 165, and Ser 196). Hence, our results expand the accessibility of OVLe's bioactive components as a promising natural source for the manufacture of physiologically active components and the creation of green biosynthesis of metal nanocrystals.

Keywords: Enterococcus faecium; Olea europaea; anti-cancer; antimicrobial; green biosynthesis; molecular docking; molecular dynamic simulation; nanocrystal.

Figure 1

(A) Olea europaea L. folium (Oleaceae) leaves; (B) Botanical Voucher Specimen, MOBOT, Tropicos.org (http://www.tropicos.org/Image/100000992, accessed on: 1 April 2022).

Figure 2

UV–Vis absorption spectra: (A) Olive leaf extract (OVLe), (B) CuO @OVLe NCs.

Figure 3

FTIR spectra: (A) Olive leaf extract (OVLe), (B) CuO @OVLe NCs.

Figure 4

XRD of CuO @OVLe NCs.

Figure 5

(A) Average particle size of prepared CuO @OVLe NCs evaluated utilising TEM (scale bar = 50 nm). (B) The size distribution of prepared CuO @OVLe NCs data is shown as the mean ± SD (n = 3).

Figure 6

(A) The shape of synthesized CuO @OVLe NCs assessed utilising SEM (scale bar = 1 µm); (B) EDX profile.

Figure 7

(A) Zone of inhibition (mm) plate with diverse concentrations 10, 15, 20, 25, and 30 mg/mL of OVLe; (B) Enterococcus faecium bacterial growth curve; (C,D) zone of inhibition (mm) plate with diverse concentrations 10, 20, and 30 mg/mL of CuO @OVLe NCs; (E) Enterococcus faecium bacterial growth curve.

Figure 8

Viability of normal WRL68 cell and cancer cells (PC3, HepG2, and MCF-7), cells treated with different concentrations of CuO @OVLe NCs. Numerical data are described as the mean ± SD (n = 3).

Figure 9

Molecular docking study of oleuropein toward the binding pocket of EFLec. (A) The surface representation of the active-site flap of EFLec with ligand, shown at the entrance of the binding pocket. (B) Amino acids involved in a hydrogen bond with oleuropein are shown as sticks. PyMol created these figures. Oleuropein (cyans), the crystal structure of EFLec (NCBI ID WP_033741131.1) (greens), and oxygen atoms are presented in red, and nitrogen atoms are shown in blue and purple dashed lines assigned to the bonding interactions. (C) Electrostatic surface of EFLec. Positive charges are in blue, negative charges are in red, and neutral charges are in purple. (D) Van der Waals and alkyl interactions with the residues of the catalytic site that are crucial for inactivating the enzyme.

Figure 10

A three-dimensional interaction scheme of oleuropein with the catalytic site of EFLec. (A) Bioavailability radar related to physicochemical properties of molecules (criteria: MW 540.51 g/mol, fraction Csp3: 0.52, TPSA < 201.67 Ų, molar refractivity: 127.28, instauration, flexibility: 0.54 < rotatable bonds < 7), and (B) Ramachandran plot for the predicted model of the docking of oleuropein.

Figure 11

(A) RMSD plot obtained for oleuropein of the protein-ligand EFLec complexes. Protein Cα and compound RMSD are shown in blue and red colour, respectively. (B) RMSF plot of the protein’s backbone atoms during the 100 ns MD simulation. Protein Cα RMSD is shown in blue.

Figure 12

Ligand torsion profile: (A) torsion and flexibility, (B) ligand torsion angles, and (C) ligand (oleuropein) property trajectory of the ligand-EFLec complex. The ligand properties fluctuated during the beginning or intermediate simulation periods, but gradually returned to equilibrium during 100 ns of simulation, indicating that ligand was stable at the active site of the protein.

Figure 13

(A) Backbone RMSD values of the Cα atoms of the protein, (B) RMSF of the backbone atoms, (C) snapshots of the docking pose of oleuropein and the total H-bond intensity at various time intervals in the 20 ns complex MD simulations with EFLec.