Novel non-specific lipid-transfer protein (TdLTP4) isolated from durum wheat: Antimicrobial activities and anti-inflammatory properties in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages

Lipid transfer proteins (LTP) are members of the family of pathogenesis-related proteins (PR-14) that play a key role in plant defense mechanisms. In this study, a novel gene TdLTP4 encoding an antifungal protein from wheat (cv. Om Rabiaa) was cloned, overexpressed in Escherichia coli BL-21 (DE3) and enriched using ammonium sulfate fractionation. The TdLTP4 fusion protein was then tested against a panel of pathogens, food-borne and spoilage bacteria and fungi in order to evaluate the antimicrobial properties. TdLTP4 was applied to 0.5 mu g/mL LPS-induced RAW 264.7 macrophages in vitro at different concentrations (5, 10, 20, 50 and 100 mu g/mL). Levels of nitric oxide (NO), pro-inflammatory cytokines interleukin (IL)-1 beta (IL-1 beta), interleukin (IL)-6 (IL-6), tumor necrosis factor (TNF-alpha) and anti-inflammatory cytokine IL-10 in the supernatant fraction were measured using enzyme-linked immunosorbent assay (ELISA). Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were detected via Western blot. The inhibition zones and minimal inhibitory concentration (MIC) values of bacterial strains were in the range of 14-26 mm and 62.5-250 mu g/mL, respectively. Moreover, a remarkable activity against several fungal strains was revealed. TdLTP4 (5-100 mu g/mL) decreased the production of NO (IC50 = 4.32 mu g/mL), IL-6 (IC50 = 11.52 mu g/mL), IL-1 beta (IC50 = 7.87 mu g/mL) and TNF-alpha (IC50 = 8.66 mu g/mL) by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. TdLTP4 could modulate the macrophages inflammatory mode by causing reduction in iNOS and COX-2. According to these findings, TdLTP4 fusion protein could be used as natural anti-inflammatory and antimicrobial agent in food preservation and human health.