Structural and biological characterization of highly purified hepta-acyl lipid A present in the lipopolysaccharide of the Salmonella enterica sv. Minnesota Re deep rough mutant strain R595

One major component of the Salmonella enterica sv. Minnesota Re deep rough mutant (strain R595) lipopolysaccharide is hepta-acyl lipid A (LA(hepta)). In a recent publication [Tanamoto K-I, Azumi S. Salmonella-type heptaacylated lipid A is inactive and acts as an antagonist of lipopolysaccharide action on human line cells. J Immunol 2000; 164: 3149-3156] the corresponding synthetic hepta-acyl lipid A (compound 516) was reported to be agonistically inactive but to rather suppress pro-inflammatory activation by the endotoxic hexa-acyl lipid A (LA(hexa), compound 506) and S-form LPS from Escherichia coli in the human macrophage-like cell lines THP-1 and U937. These results, however, were in contrast to previous findings with human mononuclear cells (hMNC) isolated from peripheral blood, in which compound 516 was found to be an agonist, expressing low, but significant, cytokine-inducing activity as compared to LA(hexa). We have investigated the structure of natural LA(hepta) from the S. enterica sv. Minnesota Re deep rough mutant strain (R595) by TLC immunoblot, MALDI-TDF mass spectrometry and NMR spectroscopy. Using these techniques, the structural identity between LA(hepta) and the synthetic compound 516 was confirmed. In corroboration of previous findings with studies employing compound 516, purified LAh(epta) was found to induce the production of TNF-alpha, IL-1beta and IL-6 in hMNC, thus displaying moderate agonistic activity. Furthermore, we showed that LA(hepta) agonistically activated nuclear translocation of NF-kappaB in THP-1 cells, thus clearly ruling out the possibility that LAh(epta) is an antagonist and that its biological activity is influenced by the type of human myeloid cells used for testing endotoxicity (hMNC versus THP-1 cells).