Physicochemical characterization and biological activity of a glycoglycerolipid from Mycoplasma fermentans

Abstract

We report a comprehensive physicochemical characterization of a glycoglycerolipid from Mycoplasma fermentans, MfGl-II, in relation to its bioactivity and compared this with the respective behaviors of phosphatidylcholine (PC) and a bacterial glycolipid, lipopolysaccharide (LPS) from deep rough mutant Salmonella minnesota strain R595. The b«a gel-to-liquid crystalline phase transition behavior of the hydrocarbon chains with T c ¼ 30°C for MfGl-II as well as for LPS exhibits high similarity between the two glycolipids. A lipopolysaccharide-binding protein (LBP)-mediated incorporation into negatively charged liposomes is observed for both glycolipids. The determination of the supramolecular aggregate structure confirms the existence of a mixed unilamellar/cubic structure for MfGl-II, similar to that observed for the lipid A moiety of LPS. The biological data clearly show that MfGl-II is able to induce cytokines such as tumor necrosis factor-a (TNF-a) in human mononuclear cells, although to a significantly lower degree than LPS. In contrast, in the Limulus amebocyte lysate test, MfGl-II is completely inactive, and in the CHO reporter cell line it does not indicate any reactivity with the Toll-like receptors TLR-2 and -4, in contrast to control lipopeptides and LPS. These data confirm the applicability of our conformational concept of endotoxicity to nonlipid A structures: an amphiphilic molecule with a nonlamellar cubic aggregate structure corresponding to a conical conformation of the single molecules and a sufficiently high negative charge density in the backbone.

A Cellular Micro-RNA, let-7i, Regulates Toll-like Receptor 4 Expression and Contributes to Cholangiocyte Immune Responses against Cryptosporidium parvum Infection

“…Before infecting cells, oocysts were excysted to release infective sporozoites as previously described (11,28). Freshly excysted sporozoites were tested for lipopolysaccharide (LPS) activity using the Limulus Amebocyte Lysate test kit (Bio-Whittaker, Walkersville, MD) as reported by others (29) and only Limulus Amebocyte Lysate-negative sporozoites were used in the study. H69 cells are SV40-transformed normal human cholangiocytes originally derived from normal liver harvested for transplant.…”

Novel quantitative electrophoretic analysis of endotoxins on microchips

“…Endotoxins were prepared in our laboratory from smoothtype bacterial strains E. coli O83 [41], Salmonella adelaide O35 [42], and E. coli 102 (isolated from a patient urine at our laboratory) and from rough strains, Salmonella minnesota R595 [43], Shigella sonnei 41, and S. sonnei 4303 [44]. The bacteria were grown in a fermentor at 377C, and then collected by centrifugation.…”

“…The O-specific chain of LPS-O83 has a repeating unit composed by glucose, galactose, Nacetyl-b-D-glucose-amine and glucuronic acid with a molar ratio of 1:2:1:1 [41], while the LPS-O35 molecules have colitose, glucose, galactose, and N-acetyl-b-D-glucose-amine in the repeating unit with a molar ratio of 2:1:1:1 [42]. The lipid-A part of LPS-595 contains one lauric, four myristic, one palmitic, and one 3-hydroxymyristic acids bound to the diglucosamine, and three KDO rings in the core (approximate molecular mass is 2.7 kDa) [43,47]. The lipid-A part of LPS-41 and LPS-4303 consists of two lauric, two myristic, and two 3-hydroxymyristic acids bound to the diglucosamine [48].…”

CE to monitor endotoxins by protein complexation

“…The molecular mass of LPS from S. minnesota R595 is ca. 2500 Da, and the structural formula is: Li-A-(3)KDO [21]. The molecular mass of LPS from S. sonnei R562H estimated from the proposed structural formula, Li-A-(3)KDO-(1)DDHep (where DDHep, D-glycero-D-manno-Hep), is about 3000 Da [26].…”

“…Endotoxins were prepared in our laboratory from two rough strains, Salmonella minnesota R595 [21] and Shigella sonnei R562H [22] and from the smooth strain Salmonella minnesota (wild-type) [23]. The bacteria were grown at 377C in a fermentor until the late logarithmic phase (about 10 h).…”