Intracellular bacterial eradication using a novel peptide in vitro

Abstract

Aim: To determine the effect of a novel antimicrobial peptide (AMP; OP145) and cell-penetrating peptide (Octa-arginine/R8) conjugate on the killing of intracellular Enterococcus faecalis, compared to OP145 and an antibiotic combination recommended for regenerative endodontic procedures.

Methodology: The biocompatible concentrations of OP145 and OP145-R8 were determined by assessing their cytotoxicity against human macrophages and red blood cells. Spatiotemporal internalization of the peptides into macrophages was investigated qualitatively and quantitatively by confocal laser scanning microscopy and flow cytometry respectively. Killing of extracellular and intracellular E. faecalis OG1RF by the peptides was determined by counting the colony-forming units (CFU). Intracellular antibacterial activity of the peptides was compared to a double antibiotic combination. Confocal microscopy was used to confirm the intracellular bacterial eradication. Significant differences between the different test groups were analysed using one-way analysis of variance. p < .05 was considered to be statistically significant.

Results: Peptides at a concentration of 7.5 μmol/L were chosen for subsequent experiments based on the results of the alamarBlue™ cell viability assay and haemolytic assay. OP145-R8 selectively internalized into lysosomal compartments and the cytosol of macrophages. Conjugation with R8 improved the internalization of OP145 into macrophages in a temporal manner (70.53% at 1 h to 77.13% at 2 h), while no temporal increase was observed for OP145 alone (60.53% at 1 h with no increase at 2 h). OP145-R8 demonstrated significantly greater extracellular and intracellular antibacterial activity compared to OP145 at all investigated time-points and concentrations (p < .05). OP145-R8 at 7.5 μmol/L eradicated intracellular E. faecalis after 2 h (3.5 log reduction compared to the control; p < .05), while the antibiotics could not reduce more than 0.5 log CFU compared to the control (p > .05). Confocal microscopy showed complete absence of E. faecalis within the OP145-R8 treated macrophages.

Conclusions: The results of this study demonstrated that the conjugation of an AMP OP145 to a cell-penetrating peptide R8 eradicated extracellular and intracellular E. faecalis OG1RF without toxic effects on the host cells.

Keywords: Enterococcus faecalis; antimicrobial peptide; bacteria; intracellular; macrophages.