GLYCAN RESIDUES ARE INVOLVED IN THE RECOGNITION AND IMMUNOGENICITY OF S-LAYER GLYCOPROTEIN FROM Lactobacillus kefiri

MALAMUD M; CARASI P; FREIRE T; SERRADELL MA

Ciudad Autónoma de Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Surface layers are (glyco)-proteinaceous cell envelopes ubiquitously found in different bacterial species, including potentially probiotic Lactobacillus kefiri. In previous studies, we showed that S-layer glycoprotein from L. kefiri CIDCA 8348 (SLP-8348) was not able to induce the activation of macrophages by itself, but it favored the LPS-induced response, since there was a significant increase in the expression of co-stimulatory molecules, IL-6 and IL-10 in comparison with LPS-stimulated cells. In this study, we aim to investigate therole of glycan residues in SLP-8348 internalization by murine macrophagesas well as in its immunogenicity. SLP-8348 was removed with 5 M LiCl and exhaustively dialyzed against PBS. Atto-647N-labeled SLP-8348 was incubated with RAW 264.7 macrophages both at 4°C (to evaluate binding) and at 37°C (to evaluate internalization), and fluorescence intensity was measured by flow cytometry. RAW264.7 cells internalized the SLP-8348 in a process that was mediated by carbohydrate-receptor interactions since it was inhibited by glucose, mannose or EGTA, a Ca+2 chelating agent. These results correlated with the recognition of SLP-8348 by ConA. Moreover, EGTA completely abrogated the effect of SLP-8348 on LPS-stimulated RAW 264.7 cells. To test the SLP-8348 immunogenicity, one dose (10 µg/mouse) of SLP-8348 or oxidized SLP-8348 (SLPOx-8348) in combination with incomplete Freund´s adjuvant was subcutaneously injected into the right flank in front of the hind leg on BALB/c mice. After ten days, cells from inguinal lymph nodes were labeled with CFSE and stimulated in vitro with SLP-8348 (10 µg/ml) for five days. Proliferation index of CD4+ T cells as well as secretion of IFN-γ were significantly lower in the group of mice treated with SLPOx-8348 respect to SLP-8348-treated mice. This is the first report showing that glycan chains are involved in the uptake of a SLP by macrophages and are crucial in the immunogenicity of this glycoprotein.