COMBINATION OF S LAYER PROTEINS FROM PATHOGENIC Clostridioides difficile AND PROBIOTIC Lentilactobacillus kefiri ON IN VITRO ACTIVATION OF MACROPHAGES

ASSANDRI MH; MALAMUD M; TREJO FM; SERRADELL MA

Congreso; LVII SAIB Meeting - XVI SAMIGE Meeting; 2021

Clostridioides difficile is a Gram-positive, anaerobic, spore-forming pathogen, and one of the leading causes of nosocomial antibiotic-associated diarrhoea (AAD) worldwide. Active immunization with surface components or proteins involved in sporulation emerges as an alternative to the antibiotic-based treatment. The S-layer is a bidimensional self-assembled (glyco)-proteinaceous envelope that covers the surface of several pathogenic and non-pathogenic bacteria. In previous works, we have shown that glycosylated SLPs from some Lentilactobacillus kefiri strains enhance the LPS-induced stimulation in both murine and human macrophages through the interaction with C-type lectin receptors. Moreover, other researchers have shown that SLP from C. difficile could act as a Toll-like receptor 4 ligand. Thus, in the search of new C. difficile antigenic targets and potential adjuvants we started to study the ability of the SLPs derived from both C. difficile ATCC 43255 and clinical isolate 117, and the SLPs of two L. kefiri strains (CIDCA 8343 and CIDCA 83111) to activate murine macrophages in vitro both alone and combined. To achieve this, L. kefiri SLP extracts (SLP-Lk) were obtained by treating bacteria with 5M LiCl, whereas two different agents were assessed to obtain C. difficile SLP (SLP-Cd): 5 M guanidine chloride and 0.2 M glycine (pH 2.2). Then, cultured RAW264.7 cell line was treated with individual SLPs or a combination of SLP-Lk + SLP-Cd at different concentrations, and secreted IL-6 after 24 h of stimulation was measured by capture ELISA. Negative controls as well as combinations of SLP-Lk + LPS were also assessed. Regarding SLP-Cd, the extraction with 0.2 M glycine showed the best performance, and two bands of approximately 48 and 38 kDa were revealed by SDS-PAGE in both strains. However, regardless the strain, SLP-Cd did not exert a strong stimulus on macrophage even when they were tested at 30 μg/ml. On the other hand, and in contrast to what has been seen in previous assays with other SLP-Lk, SLP-CIDCA 8343 and SLP-CIDCA 83111 were able to stimulate IL-6 secretion on RAW264.7 cells at concentration greater than 5 μg/ml and 10 μg/ml, respectively (P < 0.05). Interestingly, cellular activation was significantly increased (P < 0.05) after incubation with a combination of SLP-Cd from C. difficile ATCC 43255 and either SLP-CIDCA 83111 or SLP-CIDCA 8343. A similar trend was observed with the SLP-Cd from clinical isolate 117. These preliminary results suggest that the combination of both stimuli could improve the capacity of macrophages as antigen-presenting cells, which in turn might lead to a better adaptive immune response. These findings encourage us to continue to study the potential of SLP-Cd as target antigens in combination with SLP-Lk as potential adjuvants, considering the development of active immunotherapies against C. difficile AAD.