Intrinsic conformation of lipid A is responsible for agonistic and antagonistic activity

Abstract

Lipopolysaccharides (LPS, endotoxin) represent a major virulence factor of Gram-negative bacteria, which can cause septic shock in mammals, including man. The lipid anchor of LPS to the bacterial outer membrane, lipid A, exhibits a peculiar chemical structure, harbours the`endotoxic principle' of LPS and is also responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against endotoxically active LPS. By applying orientation measurements with attenuated total reflectance (ATR) infrared spectroscopy on hydrated lipid A samples, we show here that these different biological activities are directly correlated to the intrinsic conformation of lipid A. Bisphosphorylhexaacyl lipid A molecules with an asymmetric (4/2) distribution of the acyl chains linked to the diglucosamine backbone have a large tilt angle (. 45 8) of the diglucosamine backbone with respect to the membrane surface, a conical molecular shape (larger cross-section of the hydrophobic than the hydrophilic moiety), and are endotoxically highly active. Monophosphoryl hexaacyl lipid A has a smaller tilt angle, and the conical shape is less expressed in favour of a more cylindrical shape. This correlates with decreasing endotoxic activity. Pentaand tetraacyl lipid A or hexaacyl lipid A with a symmetric acyl chain distribution (3/3) have a small tilt angle (, 25 8) and a cylindrical shape and are endotoxically inactive, but may be antagonistic.Keywords: lipid A; endotoxic shape; IR ATR; agonism; antagonism.Lipopolysaccharides (LPS), as major amphiphilic components of the outer leaflet of the outer membrane of Gram-negative bacteria, exert in isolated form a variety of biological activities in mammals and are, thus, called endotoxins [1]. Chemically, LPS consists of a hydrophilic heteropolysaccharide, which is covalently linked to a hydrophobic lipid portion, termed lipid A, which anchors the molecule to the outer leaflet of the outer membrane. As free lipid A has been shown to be responsible for the biological activity of LPS in most in vitro and in vivo test systems, it has been termed the`endotoxic principle' of endotoxin [2]. The specific requirements for endotoxin to be biologically active are still only partly defined. It has been shown, however, that for the full expression of biological activity, lipid A must possess a particular chemical composition and primary structure similar to that found in enterobacterial strains. Thus, for example, lipid A from the biologically most potent LPS of the deep rough (Re) mutant strain Escherichia coli F515 consists of a b-1,6-linked d-glucosamine (GlcN) disaccharide carrying two negatively charged phosphates and six saturated fatty acids in a defined asymmetric distribution; four at the nonreducing and two at the reducing GlcN. Variation of this composition (such as a reduction of the number of charges or the number of acyl chains), a change in their distribution or degree of saturation results in a dramatic ...

Modulation of Endotoxicity of Shigella Generalized Modules for Membrane Antigens (GMMA) by Genetic Lipid A Modifications

“…Lipid A, which anchors the LPS to the outer membrane of bacteria, is the endotoxic part of LPS. The most endotoxic form of lipid A consists of a hexa-acylated glucosamine disaccharide phosphorylated at the 1 and 4′ position with acyl chains from 12 to 14 carbons in length and an asymmetric (4/2) distribution (14, 16, 17). This structure is common to Escherichia coli and Shigella (18, 19).…”

“…The total number and the length of acyl chains plus the presence of the two phosphates in positions 1 and 4′ are critical factors for full lipid A activation of human TLR4/MD-2 (14, 16, 17, 19). Changes to the structure of the lipid A, either by removal of critical components or by replacement of one of the acyl chains by a different acyl residue, affects the binding and recognition by TLR4 and results in a lower endotoxicity in vitro (14, 16).…”

Lipid A Is More than Acyl Chains

“…The role of acyl chains and head groups on the inflammatory response initiated by lipid A has been elucidated by Seydel and coworkers, who have determined its conformation and supramolecular structure (23)(24)(25)(26). Using biophysical approaches, Seydel and his group have written extensively about the relationship between the conformation of lipid A and its ability to act as an agonist and antagonist (23).…”

“…Using biophysical approaches, Seydel and his group have written extensively about the relationship between the conformation of lipid A and its ability to act as an agonist and antagonist (23). They have shown that lipid A modifications in the acyl chain number, distribution of the chains (symmetrical or asymmetrical), and head group substitution were accompanied by a change in the tilt angle of the backbone with respect to the acyl chains, which impact the conformation of the lipid A structure and, potentially, the ability to engage Toll-like receptor 4 (TRL4).…”

“…They have shown that lipid A modifications in the acyl chain number, distribution of the chains (symmetrical or asymmetrical), and head group substitution were accompanied by a change in the tilt angle of the backbone with respect to the acyl chains, which impact the conformation of the lipid A structure and, potentially, the ability to engage Toll-like receptor 4 (TRL4). They proposed that only those lipid A molecules that assume a conical shape were biologically highly active and that structures assuming a cylindrical shape were antagonistic (23). The loss of head groups causing changes in membrane integrity and the reduction in the ability of modified lipid A to interact with TLR4 most probably explain the loss of the inflammatory response and the enhanced activity of the cationic antimicrobial peptide on the organism.…”