Bacteriocin BacSp222 and Its Succinylated Forms Exhibit Proinflammatory Activities Toward Innate Immune Cells 

Abstract

Purpose The zoonotic opportunistic pathogen Staphylococcus pseudintermedius 222 produces BacSp222 – an atypical peptide exhibiting the features of a bacteriocin, a virulence factor, and a molecule modulating the host inflammatory reaction. The peptide is secreted in an unmodified form and, additionally, two forms modified posttranslationally by succinylation. This study is a comprehensive report focusing on the proinflammatory properties of such molecules. Methods The study was performed on mouse monocyte/macrophage-like and endothelial cell lines as well as human neutrophils. The following peptides were studied: BacSp222, its succinylated forms, the form deprived of formylated methionine, and a reference bacteriocin – nisin. The measurements of the nitric oxide (NO) level, induced NO synthase (iNOS) expression, the profile of secreted cytokines, NF-kappa-B activation, reactive oxygen species (ROS) biosynthesis, and the formation of extracellular traps were conducted to evaluate the proinflammatory activity of the studied peptides. Results BacSp222 and its succinylated forms effectively induced NO production and iNOS expression when combined with IFN-gamma in macrophage-like cells. All natural BacSp222 forms used alone or with IFN-gamma stimulated the production of TNF-alpha, MCP-1, and IL-1-alpha, while the co-stimulation with IFN-gamma increased IL-10 and IL-27. Upregulated TNF-alpha secretion observed after BacSp222 exposition resulted from increased expression but not from membrane TNF-alpha proteolysis. In neutrophils, all forms of bacteriocin upregulated IL-8, but did not induce ROS production or NETs formation. In all experiments, the activities of deformylated bacteriocin were lower or unequivocal in comparison to other forms of the peptide. Conclusion All naturally secreted forms of BacSp222 exhibit proinflammatory activity against monocyte-macrophage cells and neutrophils, confirming that the biological role of BacSp222 goes beyond bactericidal and cytotoxic effects. The atypical posttranslational modification (succinylation) does not diminish its immunomodulatory activity in contrast to the lower antibacterial potential or cytotoxicity of such modified form established in previous studies.

BacSp222 bacteriocin as a novel ligand for TLR2/TLR6 heterodimer

“…BacSp222 bacteriocin, its succinylated form (suc-K20-BacSp222), as well as bacteriocin deprived of Nterminal formyl-methionine (-fM-BacSp222) were obtained and analyzed strictly as described in our previous work [15]. As is presented in the above-cited study, the purity (over 99%), homogeneity, and identity of the peptides were checked by an analytical reversed-phase high-pressure liquid chromatography (RP-HPLC) and mass spectrometry.…”

“…As we have shown above, all tested forms of BacSp222 bacteriocin were able to activate the human TLR2 receptor. Since our previously published results demonstrated that BacSp222 induced TNF expression in murine macrophage-like cell line [15], we have decided to verify if TLR2 antagonists, such as sparstolonin B or TL2-C29 [22,23], affect the TNF production by bacteriocin-stimulated RAW 264.7 and P388.D1 cells. The cells were pre-treated with mentioned antagonists for 30 minutes and then stimulated for 6 hours with speci c BacSp222 bacteriocin or LTA (the ligand for TLR2 and a positive control of the test).…”

“…Next, the media were collected for TNF concentration measurement using the ELISA test (ELISA MAX™ Standard Set Mouse TNF; Biolegend, San Diego, CA, USA) according to the protocol provided by the manufacturer. The viability of the cells was determined and analysed using a MTT assay as described previously [15]. The absorbance was measured at 450 nm for ELISA and 545 nm for MTT assays using the Synergy H1 Hybrid plate reader controlled by Gene5 version 2.00.18 software (BIOTEK Instruments, Winooski, VT, USA).…”

“…In murine macrophage-like cell line, it resulted in increased secretion of TNF, MCP-1, and IL-1α, as well as enhanced nitric oxide (NO) production by inducible NO synthase (iNOS). In human neutrophils, BacSp222 upregulated IL-8 production but, on the other hand, did not induce reactive oxygen species (ROS) production nor neutrophil extracellular traps (NETs) formation [15].…”

“…The present study concerns the identi cation of cellular receptors responsible for sensing BacSp222 by eukaryotic cells. We focused on two particular classes of receptors, formyl peptide receptors (FPRs) and Toll-like receptors (TLRs), present on various innate immune cells and able to detect bacterial-derived pathogen-associated molecular patterns (PAMPs), as well as to induce in ammatory response [15,16].…”