Surface Layer-Associated Protein on Nanoparticles from Lactobacillus helveticus Enhances the Response to LPS in RAW264.7 Cells.

Lactic acid bacteria that persist in the gastrointestinal tract contribute not only to nutrient metabolism but also to the regulation of immune responses. Accordingly, these microorganisms and their components are classified as probiotics when administered in adequate amounts and shown to confer health benefits on the host. However, the mechanisms by which they interact with host cells remain poorly understood. Extracellular vesicles (EVs) have been shown to modulate functions in recipient cells, including both mammalian cells and microorganisms. Surface-layer associated proteins (SLAPs) are preferentially produced by Gram-positive syntrophic bacteria and are secreted to associate with the cell envelope. The dysfunction of SLAPs leads to disruption of the cell envelope, cell division, and cell-cell communication. In this study, we identified SLAP in extracellular vesicle-like nanoparticles (EV-LNPs) derived from Lactobacillus helveticus strain GIF001 (closed to strain VHProbi Y21) and observed enhanced immune responses-including nitric oxide and interleukin-6 production-in RAW264.7 cells when SLAP was added to the culture medium, either in the form of EV-LNPs or as a purified protein. Notably, L. helveticus EV-LNPs were internalized within 3 h and enhanced NF-κB signaling in response to lipopolysaccharide. Intracellular overexpression of SLAP led to increased activity of the inducible nitric oxide synthase promoter about 1.5 times, whereas deletion of the SLAP domain abolished this enhancement. Incubation of RAW264.7 cells with EV-LNPs or SLAP enhanced the uptake of Escherichia coli. These results suggest that EV-LNPs from L. helveticus may upregulate immune responses by activating host cell communication via SLAP.