Plasma endotoxin and lipopolysaccharide-binding protein (LBP) levels were measured in a group of 253 patients at the onset of severe sepsis and/or septic shock. Endotoxin levels were significantly greater than control levels (n=33; mean +/- SD, 5.1+/-7.3 pg/mL) in 78.3% of patients. Median endotoxin levels in patients with sepsis were 300 pg/mL (25%-75% interquartile range, 110-726 pg/mL). LBP levels were elevated in 97% of patients compared with normal control values of 4.1+/-1.65 microgram/mL. Median LBP levels in patients with sepsis were 31.2 microgram/mL (interquartile range, 22.5-47.7 microgram/mL). Median endotoxin levels at study entry were more highly elevated (515 vs. 230 pg/mL; P<.01), and LBP levels were less highly elevated (28.0 vs. 33.2 microgram/mL; P<.05) in nonsurvivors than survivors over the 28-day study period. No correlation was found between endotoxin and LBP levels. The quantitative level of both endotoxin and LBP may have prognostic significance in patients with severe sepsis.

Bactericidal/permeability-increasing protein (BPI), a potent antimicrobial protein of 456 residues, binds to and neutralizes lipopolysaccharides from the outer membrane of Gram-negative bacteria. At a resolution of 2.4 angstroms, the crystal structure of human BPI shows a boomerang-shaped molecule formed by two similar domains. Two apolar pockets on the concave surface of the boomerang each bind a molecule of phosphatidylcholine, primarily by interacting with their acyl chains; this suggests that the pockets may also bind the acyl chains of lipopolysaccharide. As a model for the related plasma lipid transfer proteins, BPI illuminates a mechanism of lipid transfer for this protein family.

Exotoxin A of Pseudomonas aeruginosa is a secreted bacterial toxin capable of translocating a catalytic domain into mammalian cells and inhibiting protein synthesis by the ADP-ribosylation of cellular elongation factor 2. The protein is a single polypeptide chain of 613 amino acids. The x-ray crystallographic structure of exotoxin A, determined to 3.0-A resolution, shows the following: an amino-terminal domain, composed primarily of antiparallel a8-structure and comprising approximately half of the molecule; a middle domain composed of a-helices; and a carboxyl-terminal domain comprising approximately one-third of the molecule. The carboxyl-terminal domain is the ADP-ribosyltransferase of the toxin. The other two domains are presumably involved in cell receptor binding and membrane translocation.Exotoxin A of Pseudomonas aeruginosa is one member of a family of secreted bacterial toxins that are capable of covalently modifying specific target proteins within mammalian cells (1). Included in this family are the exotoxins of Corynebacterium diphtheriae (diphtheria toxin) and Vibrio cholerae (cholera toxin), Escherichia coli heat-labile toxin (LT), and exotoxins of Shigella dysenteria (shiga toxin) and Bacillus anthracis (anthrax toxins) as well as exotoxin A (2). Despite their diversity in size, subunit composition, cell specificity, and enzymatic activity, these toxins appear to share a similar multistep mechanism in which (i) the toxin binds to a receptor on the membrane surface of a target cell; (ii) the catalytic domain of the toxin is translocated into, or at a minimum into contact with, the cell cytoplasm; (iii) the catalytic moiety is then able to modify its target substrate. The toxins thus must have a receptor binding activity, a membrane translocation mechanism, and an enzymatic domain. It is characteristic that the receptor binding function and the enzymatic activity reside in separate structural components of the molecules, in separate subunits of an oligomer (cholera toxin, LT, shiga toxin) (3-5), in separate proteins (anthrax system) (6), or within a single monomeric polypeptide (diphtheria toxin, exotoxin A) (7,8).Several of the toxins (cholera toxin, LT, diphtheria toxin, and exotoxin A) catalyze transfer of the ADP-ribose moiety of oxidized nicotinamide adenine dinucleotide (NAD+) to target substrates (9-12). Diphtheria toxin and exotoxin A specifically ADP-ribosylate a modified histidine (diphthamide) of protein synthesis elongation factor 2, thereby inactivating the elongation factor and terminating peptide chain elongation in a target cell (13).Several intriguing mechanistic questions arise: (i) What are the mechanisms of membrane translocation by which the toxic factors enter the target cell cytoplasm? (ii) How is the membrane translocation and enzymic activation process controlled during intoxication? (iii) What is the mechanism of the ADP-ribosyltransferase reaction? Little structural information is available for members of this class of bacterial toxins. Crystals suitable for high re...

The lipopolysaccharide (LPS)-binding protein (LBP) has a concentration-dependent dual role in the pathogenesis of gram-negative sepsis: low concentrations of LBP enhance the LPS-induced activation of mononuclear cells (MNC), whereas the acute-phase rise in LBP concentrations inhibits LPS-induced cellular stimulation. In stimulation experiments, we have found that LBP mediates the LPS-induced cytokine release from MNC even under serum-free conditions. In biophysical experiments we demonstrated that LBP binds and intercalates into lipid membranes, amplified by negative charges of the latter, and that intercalated LBP can mediate the CD14-independent intercalation of LPS into membranes in a lipid-specific and temperaturedependent manner. In contrast, prior complexation of LBP and LPS inhibited binding of these complexes to membranes due to different binding of LBP to LPS or phospholipids. This results in a neutralization of LPS and, therefore, to a reduced production of tumor necrosis factor by MNC. We propose that LBP is not only present as a soluble protein in the serum but may also be incorporated as a transmembrane protein in the cytoplasmic membrane of MNC and that the interaction of LPS with membrane-associated LBP may be an important step in LBP-mediated activation of MNC, whereas LBP-LPS complexation in the serum leads to a neutralization of LPS.Human lipopolysaccharide (LPS)-binding protein (LBP) is a serum glycoprotein belonging to a family of lipid-binding proteins which includes bactericidal/permeability-increasing protein (BPI), phospholipid ester transfer protein, and cholesterol ester transfer protein (1,18,36). It consists of 456 amino acid residues preceded by a hydrophobic signal sequence of 25 residues (31). LBP is synthesized by hepatocytes (26) and intestinal epithelial cells (42) and is present in normal serum at concentrations of 5 to 10 g/ml, rising up to 200 g/ml 24 h after induction of an acute-phase response (35). This rise in LBP levels is caused by transcriptional activation of the LBP gene mediated by interleukin-1 (IL-1) and . LBP has a concentration-dependent dual role: low concentrations of LBP enhance the LPS-induced activation of mononuclear cells (MNC), whereas the acute-phase rise in LBP concentrations inhibits LPS-induced cellular stimulation (20). LBP binds a variety of LPS (endotoxin) chemotypes from rough and smooth strains of gram-negative bacteria and even lipid A, the lipid moiety of LPS (37, 38). The LPS molecules, components of the outer membrane of gram-negative bacteria, are important mediators in the pathogenesis of gram-negative sepsis and septic shock (25). Because the lipid A moiety has been shown to be responsible for the biological activity of LPS in most in vivo and in vitro test systems, it has been termed the endotoxic principle of LPS (27).LPSs activate monocytes and macrophages to secrete inflammatory cytokines (tumor necrosis factor alpha [TNF-␣] and IL-1, etc.) and other potent mediators (32) by an intracellular signal amplification pathway. These mediato...