BacSp222 bacteriocin as a novel ligand for TLR2/TLR6 heterodimer

Objective and designBacSp222 bacteriocin is a bactericidal and proinflammatory peptide stimulating immune cells to produce selected cytokines and NO in NF-kappa B dependent manner. This study aims to identify the receptor which mediates this activity.MethodsWe applied fluorescently labeled BacSp222 and a confocal microscopy imaging to analyze the direct interaction of the bacteriocin with the cells. Reporter HEK-Blue cells overexpressing human toll-like receptors (TLR2, TLR4, TLR5 or TLR2/TLR1 and TLR2/TLR6 heterodimers) were stimulated with BacSp222, and then the activity of NF-kappa B-dependent secreted embryonic alkaline phosphatase (SEAP) was measured. In turn, formylated peptide receptor (FPR) or TLR2 antagonists were used to verify bacteriocin-stimulated TNF production by murine monocyte-macrophage cell lines.ResultsBacSp222 undergoes internalization into cells without disturbing the cell membrane. FPR antagonists do not affect TNF produced by BacSp222-stimulated murine macrophage-like cells. In contrast, BacSp222 stimulates NF-kappa B activation in HEK-Blue overexpressing TLR2 or TLR2/TLR6 heterodimer, but not TLR2/TLR1, TLR4 or TLR5 receptors. Moreover, TLR2-specific antagonists inhibit NF-kappa B signaling in BacSp222-stimulated HEK-Blue TLR2/TLR6 cells and reduce TNF release by BacSp222-treated RAW 264.7 and P388.D1.ConclusionsBacSp222 is a novel ligand for TLR2/TLR6 heterodimer. By binding TLR complex the bacteriocin undergoes internalization, inducing proinflammatory signaling that employs MyD88 and NF-kappa B pathways.

Automated summary

The objective of this study is to identify the receptor that mediates the activity of BacSp222 bacteriocin. Through fluorescent labeling and confocal microscopy, it was found that BacSp222 interacts directly with cells. The results show that BacSp222 binds to TLR2/TLR6 heterodimers and induces proinflammatory signaling through MyD88 and NF-kappa B pathways.