Biological activity of synthetic heptaacyl lipid A representing a component of Salmonella minnesota R595 lipid A

Abstract

A synthetic lipid A (preparation 516), containing seven acyl groups and representing one component of natural free lipid A of Salmonella minnesota R595, has been investigated for biological activity in a number of endotoxin test systems. It was found that the synthetic preparation was, in typical in vivo endotoxin tests (lethality, pyrogenicity, Shwartzman reactivity) as well as in its antigenicity and macrophage activation capacity, significantly less active than natural Salmonella lipid A. However, in other in vitro assay systems (B-cell mitogenicity, complement activation, timulus amoebocyte lyzate gelation) it expressed similar activity as Salmonella lipid A.Lipid A represents the endotoxic principle of lipopolysaccharides from which it can be released and isolated by mild acid treatment. Lipid A (in its free or bound form) consists of a heterogeneous mixture of structurally closely related molecular species. The main component of free lipid A of Escherichia coli F515 (Re mutant) represents a hexaacyl lipid A containing the bisphosphorylated glucosamine disaccharide backbone substituted with six acyl residues, i.e. one each of dodecanoic and tetradecanoic acid and four of (R)-3-hydroxytetradecanoic acid, the fatty acids being located at specific positions [l] (see Fig. 1 A). This hexaacyl E. colilipid A has recently been chemically synthesized [2] and the synthetic material, preparation 506, has been shown to be endotoxically fully active, its biological potency being indistinguishable from that of natural E. coli lipid A [3 -51.Analytical stuhes performed with free lipid A from Salmonella minnesota R595 (Re mutant) led to the recognition of a basically identical structure except that it contains an additional hexadecanoic acid (Fig. 1 B). It was found, however, that this fatty acid is not present in stoichiometric amounts [l] and that the heptaacyl lipid A species represents, depending on the bacterial batch, only a fraction of about 20-70% of the microheterogeneous lipid A preparation. Other molecules in the mixture contain smaller numbers of acyl residues and hence resemble or are identical to the E. coli lipid A [6]. Recently, that molecular species of Salmonella lipid A which contains seven acyl residues (heptaacyl lipid A), has been chemically synthesized and termed preparation 516 (Shiba et al., unpublished MATERIAL AND METHODS Bacterial lipid AFree lipid A from S. minnesota R595 (Re mutant) and E. coli F515 (Re mutant) was prepared by treatment of the respective Iipopolysaccharide with 0.1 M acetate buffer (pH 4.4, 100°C) according to [7]. The preparations were freed of low-molecular-mass cations as described earlier and used in the triethylammonium salt form [S]. Synthetic lipid AThe synthesis of hexaacyl lipid A (preparation 506) has been described [2]. The heptaacyl lipid A (preparation 516) was prepared according to the same synthetic strategy by coupling of the two monosaccharide components, i.e. 2 -glucopyranoside to yield the 1,6-linked disaccharide (T. Shiba, unpublished). For conversion in...

R‐form LPS, the master key to the activation ofTLR4/MD‐2‐positive cells

“…R-form LPS from S. minnesota mutants: R 595 (Re), R3 (Rd), R7 (Rd), R5 (Rc), R345 (Rb) and R60 (Ra) were extracted and purified as described [62,64]. Lipid A from S. minnesota R595 was prepared as described [65]. Re-form LPS of E. coli, serotype 515 (liquid) and lipid A from E. coli, serotype 515 (liquid) were from Alexis Deutschland GmbH (Grünberg, Germany).…”

Salmonella-Type Heptaacylated Lipid A Is Inactive and Acts as an Antagonist of Lipopolysaccharide Action on Human Line Cells

“…Both of these lipid As, the Salmonella-type heptaacylated and E. coli-type hexaacylated forms, had been successfully synthesized chemically in the past (12)(13)(14), and their biological properties had been carefully tested by many investigators (15)(16)(17)(18)(19)(20). The Salmonella-type lipid A had been demonstrated to be very active in mice and rabbits and subsequently had been thought to be a typical lipid A (18 -20) along with that from E. coli.…”

“…Escherichia coli-and Salmonella-type lipid A have the most typical lipid A structures so far defined, and both have been synthesized chemically (12)(13)(14) and well characterized biologically by many investigators (15)(16)(17)(18)(19)(20). The chemical structure of these lipid As consists of a hexa-and heptaacylated diglucosamine bearing six and seven fatty acids, respectively, as shown in Fig.…”

Comparison of lipids A of several Salmonella and Escherichia strains by 252Cf plasma desorption mass spectrometry

“…That it is heterogeneous can be demonstrated by TLC (4,9). Comparison by TLC of preparations obtained from different batches of this strain showed that they varied both quantitatively and qualitatively (not shown).…”

“…induced us to examine a broader sample of these lipids A. The use in current literature (7,10,28) of the terms "Escherichia-" and "Salmonella-type" lipid A to designate synthetic compounds has led to the idea that these types correspond to the major molecular species present in any given preparation, although the Salmonella type was clearly presented as a component of Salmonella Minnesota R595 lipid A (9). The examination by PDMS of lipid A preparations isolated from several strains of these genera (8 Fatty acids were analyzed by hydrolyzing the endotoxins in 4 M HCl for 2 h at 100'C, neutralization followed by hydrolysis with 2 M NaOH for 2 h at 100'C (12), extraction with hexane, methylation by diazomethane, and identification by retention time on a BP10 gas chromatography column (Scientific Glass Engineering, North Melbourne, Australia) in a temperature gradient of 160 to 240'C at 40C/min.…”