Extracellular products from virulent strain of Edwardsiella tarda stimulate mouse macrophages (RAW264.7) to produce nitric oxide (NO) and tumor necrosis factor (TNF)-α

Abstract

We have previously found that high virulent strain (NUF251) of Edwardsiella tarda, but not low virulent strain (NUF194), was able to survive and multiply within Japanese flounder ( Paralichthys olivaceus) peritoneal macrophages. Further studies demonstrated that NUF251 induced much higher levels of NO and TNF-α productions than NUF194 in both Japanese flounder peritoneal macrophages and mouse macrophage cell line RAW264.7. In this study, we examined the effects of extracellular products (ECP) from two strains of E. tarda on RAW264.7 cells in terms of the induction of NO and TNF-α. ECP from NUF251 stimulated RAW264.7 cells to induce NO production in a concentration-dependent manner. The activity of NUF251-ECP completely disappeared by heat-treatment (at 100 °C for 5 min), but it could not be removed by dialysis. Polymyxin B, an endotoxin inhibitor, had no effect on NUF251-ECP-induced NO production. These results suggest that active agents in NUF251-ECP responsible for NO induction may be heat-labile high molecular weight substances rather than the cell wall derived endotoxin like substances. Since NO synthase (NOS) inhibitor, l-NAME, suppressed NUF251-ECP-induced NO production, inducible NO synthase (iNOS) in RAW264.7 cells may be a main source of NO. Furthermore, NUF251-ECP-induced high level of TNF-α secretion from RAW264.7 cells. Both NO and TNF-α productions induced by NUF251-ECP were significantly blocked by a JNK inhibitor. In contrast to NUF251-ECP, no significant activities of NUF194-ECP to induce NO and TNF-α productions were detected. SDS-PAGE and subsequent proteomic analysis of ECP from both strains suggested that NUF251-specific protein, which has sequence homology with flagellin, is present in NUF251-ECP as a main component. Our results suggest that the high virulent strain (NUF251) of E. tarda may specifically produce an exotoxin capable of inducing high levels of NO and TNF-α from macrophages through the activation of JNK system, and most probable candidate for such exotoxin might be a flagellin-like protein.