Structural investigations into the interaction of hemoglobin and part structures with bacterial endotoxins.

An understanding of details of the interaction mechanisms of bacterial endotoxins (lipopolysaccharide, LPS) with the oxygen transport protein hemoglobin is still lacking, despite its high biological relevance. Here, a biophysical investigation into the endotoxin:hemoglobin interaction is presented which comprises the use of various rough mutant LPS as well as free lipid A; in addition to the complete hemoglobin molecule from fetal sheep extract, also the partial structure alpha-chain and the heme-free sample are studied. The investigations comprise the determination of the gel-to-liquid crystalline phase behaviour of the acyl chains of LPS, the ultrastructure (type of aggregate structure and morphology) of the endotoxins, and the incorporation of the hemoglobins into artificial immune cell membranes and into LPS. Our data suggest a model for the interaction between Hb and LPS in which hemoglobins do not react strongly with the hydrophilic or with the hydrophobic moiety of LPS, but with the complete endotoxin aggregate. Hb is able to incorporate into LPS with the longitudinal direction parallel to the lipid A double-layer. Although this does not lead to a strong disturbance of the LPS acyl chain packing, the change of the curvature leads to a slightly conical molecular shape with a change of the three-dimensional arrangement from unilamellar into cubic LPS aggregates. Our previous results show that cubic LPS structures exhibit strong endotoxic activity. The property of Hb on the physical state of LPS described here may explain the observation of an increase in LPS-mediating endotoxicity due to the action of Hb.

Influence of commercial sanitizers on lipopolysaccharide production by Salmonella Enteritidis ATCC 13076.

The effect of typical sanitizers on the composition and toxicity of lipopolysaccharides (LPSs) produced by Salmonella Enteritidis ATCC 13076 was analyzed. Salmonella Enteritidis was propagated up to the late exponential phase in the presence of commercial sanitizing solutions. LPS was extracted and derivatized with trifluoroacetylation, and gas chromatography-mass spectrometry analysis and the chromogenic Limulus amoebocyte lysate assay were used to assess the ultrastructure and toxicity of the LPS. The viability and debris formation during growth were evaluated to verify the bactericidal and bacteriostatic effects of the sanitizers and to assess sanitizer effects on LPS formation. The LPSs produced were quantified at 1.7 x 10(4), 1.2 x 10(4), 3.6 x 10(3), and 9.6 x 10(4) [KDO] x OD(620nm)(-1) for the controls and the organisms grown in the presence of a chlorinated sanitizer, a heavy-duty alkaline cleaner, and a phenolic hand wash solution, respectively. In response to these treatments, the short-chain polysaccharide fractions of the LPSs in the Salmonella Enteritidis cells increased. This finding suggests that this organism increases the low-molecular-weight fraction of the LPS in relation to the high-molecular-weight fraction to survive these unfavorable conditions. The cumulative change in the LPS in response to the sanitizers influenced the toxicity of the LPS; however, this change could not be related to an individual compound within any of the assessed fractions.

The influence of sanitizers on the lipopolysaccharide composition of Escherichia coli O111.

This study focused on the influence of typical sanitizers on the composition of the lipopolysaccharides (LPS) produced by the verocytotoxin-producing (VTEC) Escherichia coli O111. We also aimed to cast light on the applicability of O-antigen-based serotyping and endotoxin based Limulus Amebocyte Lysate (LAL) assays applied in the food industry for the identification and quantification of Gram-negative bacteria. E. coli O111 was propagated in the presence of three typical commercially applied sanitizing solutions that included a Clean in Place (CIP) chlorinated sanitizer (bacteriocidal), heavy-duty alkaline sanitizer (bacteriocidal) and a phenolic hand wash solution (bacteriostatic). After the required growth phase was reached the LPS from both the intact cells and debris was extracted and methanolysed followed by trifluoroacetylation. Subsequently GC-MS analysis and the chromogenic LAL assay were applied to assess both the ultra-structure and the toxicity of the extracted LPS. The viability and debris formation during growth was also evaluated to verify the bacteriocidial and static effect of the applied sanitizers as well as to assess its relationship with LPS formation. The total LPS produced was quantified at 1.3 x 10(6) [KDO] x OD(620 nm)(-1) for the control samples, 6.5 x 10(3) [KDO] x OD(620 nm)(-1) for E. coli grown in the presence of CIP chlorinated sanitizer and 2.1 x 10(5) and 2.85 x 10(6) [KDO] x OD(620 nm)(-1) for the organisms grown in the presence of heavy-duty alkaline sanitizer and phenolic hand wash solution respectively (KDO = 2-keto-3-deoxy-octulosonic acid). A negative correlation (gamma(2)= -0.880) between the [KDO] and Delta viability was evident and indicated that E. coli O111 responds to factors that hinder viability by producing more LPS in its outer membrane. Subsequent assessment of the LPS ultra-structure revealed a definite change in both the total assessed saccharide and lipid fractions. The cumulative change of the LPS in response to the sanitizers further appeared to influence the toxicity of the LPS as the latter change could not be related to an individual compound within any of the assessed fractions. This emphasised the fact that the quantity of LPS obtained from E. coli O111 in this study, did not seem to determine the toxicity of the organism. From the results we further propose a coefficient that could be applied to describe the response of E. coli O111 LPS to sanitizers and caution against the application of serotyping (based on the O-antigen) and the LAL assay to quantify and identify E. coli O111 obtained from food strata where the possibility of sanitizer contamination exists.

Characterization of Brucella abortus lipopolysaccharide macrodomains as mega rafts.

The lipopolysaccharides (LPS) of intracellular Proteobacteria such as Brucella, Chlamydia, Legionella and Rickettsia, have properties distinct from enterobacterial LPSs. These properties include deficient LPS induction of host cell activation, low endotoxicity and resistance to macrophage degradation. Together these constitute key virulence mechanisms for intracellular survival and replication. We previously demonstrated that B. abortus LPS captured by macrophages was recycled back to the plasma membrane where it was found associated with macrodomains. Furthermore, this LPS interferes with the MHC class II (MHC-II) presentation of peptides to specific T cell hybridomas. Here, we characterized the Brucella LPS macrodomains by microscopy and biochemistry approaches. We show for the first time that LPS macrodomains act as detergent resistant membranes (DRMs), segregating several lipid-raft components, LPS-binding proteins and MHC-II molecules. Brucella LPS macrodomains remain intact for several months in macrophages and are resistant to the disruptive effects of methyl beta-cyclodextrin. Fluorescent anisotropy measurements show that B. abortus LPS is responsible for the formation of rigid surface membrane complexes. In addition, relocalization of MHC-II molecules is observed in these structures. The effects of B. abortus LPS on membrane properties could be responsible for pathogenic effects such as the inhibition of MHC-II-dependent antigen presentation.

Highly ordered supra-molecular organization of the mycobacterial lipoarabinomannans in solution. Evidence of a relationship between supra-molecular organization and biological activity.

The complex mycobacterial mannosylated lipoarabinomannans (ManLAMs) are currently considered to be the major virulence factors of the pathogenic Mycobacterium tuberculosis. The recognition and the interaction of ManLAMs with immune system receptors have been shown to promote M.tuberculosis phagocytosis but also to down-regulate the bactericidal immune response of the host in favor of the survival of the pathogenic bacilli. To date these original biological activities were mainly associated to the presence of mannose residues capping the non-reducing ends of the ramified polysaccharide moiety of these complex lipoglycans. However, we demonstrated recently that the molecular recognition of ManLAM terminal mannose units by human pulmonary surfactant protein A (hSP-A) carbohydrate recognition domains depends on the presence of the lipid moiety of the ManLAMs as proposed by Sidobre et al. in 2002. Thus, we investigated the putative role of the ManLAM aglycon moiety. The data presented here, indicate that the hydrophobic aglycon part of ManLAM is associated to a characteristic concentration-dependent supra-molecular organization of these complex molecules. Furthermore, we observed that the deacylated ManLAMs or the lipid-free mannosylated arabinomannans, which do not exhibit characteristic ManLAM activities, do not display this supra-molecular organization. These observations strongly suggest that the ManLAMs immunomodulatory activities might be associated to their particular organization. Finally, the determination of the critical micellar concentration of ManLAMs obviously supports the notion that this supra-molecular organization may be responsible for the specific biological activities of these complex molecules.

Diphosphoryl lipid A from Rhodobacter sphaeroides blocks the binding and internalization of lipopolysaccharide in RAW 264.7 cells.

Diphosphoryl lipid A derived from the nontoxic LPS of Rhodobacter sphaeroides (RsDPLA) has been shown to be a powerful LPS antagonist in both human and murine cell lines. In addition, RsDPLA also can protect mice against the lethal effects of toxic LPS. In this study, we complexed both the deep rough LPS from Escherichia coli D31 m4 (ReLPS) and RsDPLA with 5- and 30-nm colloidal gold and compared their binding to the RAW 264.7 cell line by electron microscopy. Both ReLPS and RsDPLA bound to the cells with the following observations. First, binding studies revealed that pretreatment with RsDPLA completely blocked the binding and thus internalization of ReLPS-gold conjugates to these cells at both 37 degrees C and 4 degrees C. Second, ReLPS was internalized via micropinocytosis (noncoated plasma membrane invaginations) involving formation of caveolae-like structures and leading to the formation of micropinocytotic vesicles, macropinocytosis (or phagocytosis), formation of clathrin-coated pits (receptor mediated), and penetration through plasma membrane into cytoplasm. Third, in contrast, RsDPLA was internalized predominantly via macropinocytosis. These studies show for the first time that RsDPLA blocks the binding and thus internalization of LPS as observed by scanning and transmission electron microscopy.

Hexagonal assembly of the magnesium salt of an R-form lipopolysaccharide from Klebsiella pneumoniae: its lowered stability compared with original non-electrodialyzed preparation.

The magnesium salt of R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-:K1-) that was prepared after the removal of cationic materials by electrodialysis formed essentially the same ordered hexagonal lattice structure with a lattice constant of 14 to 15 nm as the original non-electrodialyzed preparation of the R-form LPS. When the magnesium salt was suspended in 50 mM glycine buffer or Tris buffer at pH 1.4 to 9.5 and kept at 4 C for 24 hr, its content of Mg was markedly decreased, and its hexagonal lattice structure was changed to a swollen hexagonal lattice structure with extended lattice constants at pH 1.4 and to a loose mesh-like structure at pH 3.0 or higher. In the original non-electrodialyzed preparation of the R-form LPS, the release of Mg and disintegration of the hexagonal lattice structure did not occur by suspending in buffers at pH 1.4 to 8.5 at 4 C for 24 hr, but occurred only at pH 9.0 or higher. The results suggest that organic cations that can be removed by electrodialysis play some part in tight binding to Mg2+ and in stabilizing the ordered hexagonal assembly of the R-form LPS.

Molecular structure of bacterial endotoxin (Escherichia coli Re lipopolysaccharide): implications for formation of a novel heterogeneous lattice structure.

Analyses of crystals of Escherichia coli Re lipopolysaccharide (LPS) formed after storage in 1% triethylamine indicate that the LPS molecules are assembled to form a monolayered structure consisting of a novel heterogeneous lattice structure, the greater part of which is occupied by one kind of lattice (lattice I), corresponding to the acyl chain portion of lipid A, and the remainder is occupied by the other kind of lattice (lattice II), corresponding to the 3-deoxy-Dmanno-octulosonic acid (dOclA) dimer and the N-acetylglucosamine disaccharide of lipid A. X-ray diffraction reveals that the type of cell is monoclinic (a = 5.53 A, b = 27.2 A, c = 6.47 A, alpha = 90 degrees, beta = 125.8 degrees, gamma = 90 degrees ). Atomic force microscopy shows that crystals consist of multiple layers; the thickness of a layer corresponds to the b-axis value, and two types of surface topographies are visualized. One, regarded as the view onto the acyl chain ends, is two-dimensional arrays of oval bodies that constitute the lattice, with the lattice constants corresponding to the a- and c-axes and the angle of beta (lattice I). The other, regarded as the view onto the dOclA dimers, is two-dimensional arrays of dromedary-back-like bodies that constitute the lattice with axes of 9.0 and 10.7 A and the angle of 65 degrees formed by both axes (lattice II). Based on these results, we present the molecular model of E. coli Re LPS.

Disintegration of Mg2+ -induced hexagonal assembly of an R-form lipopolysaccharide from Klebsiella pneumoniae by treatment with CaCl2.

R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-: K1-), which was precipitated by the addition of 2 volumes of ethanol containing 10 mM MgCl2 for the purification process, ultrastructurally exhibited membrane pieces consisting of an ordered hexagonal lattice structure with a lattice constant of 14 to 15 nm. When the R-form LPS was suspended in 50 mM tris (hydroxymethyl) aminomethane buffer (at pH 8.5) containing 1 mM or higher concentrations of CaCl2 and kept at 4 C for 10 hr, the ordered hexagonal lattice structure of the R-form LPS was disintegrated and changed to an irregular rough, mesh-like structure. By treatment with CaCl2, the content of Mg in the LPS was markedly decreased, and conversely, the content of Ca was increased to a level depending upon the concentration of CaCl2. Results indicate that the addition of CaCl2 to suspensions of the Mg-bound R-form LPS result in a tighter binding of Ca2+ to the R-form LPS and the release of Mg2+ from the R-form LPS, and as a consequence, destroys the Mg2+ -induced ordered hexagonal lattice structure of the R-form LPS.

Direct visualization of gram-negative bacterial lipopolysaccharide self-assembly.

Bacterial lipopolysaccharides (LPS) are outer cell wall components of gram-negative bacteria that may cause septic shock in mammals. The exact morphology of LPS when interacting with macromolecular complexes of the septic shock pathway in blood is still uncertain. Here, the geometry and morphology of hydrated bacterial LPS, dispersed in solution, at and below its the critical aggregate concentration, were directly examined by tapping mode atomic force microscopy (TMAFM) and dynamic light scattering. High-resolution phase-shift TMAFM images of hydrated LPS of Salmonella minnesota Re595, Salmonella typhimurium, and Escherichia coli 0111:B4 adsorbed on mica surfaces unveiled nanosized lipidic particles with a species-specific organization. The complex hydrodynamic geometry exhibited by LPS in dilute suspensions may have consequences for the interpretation of LPS biological activity in the host immune response.

Bacterial lipopolysaccharide can enter monocytes via two CD14-dependent pathways.

Host recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes. We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchored CD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonic medium obliterated coated pits and blocked 125I-labeled transferrin internalization, but failed to inhibit CD14-mediated internalization of [3H]LPS monomers or aggregates. Immunogold electron microscope analysis found that CD14-bound LPS moved principally into noncoated structures (mostly tubular invaginations, intracellular tubules, and vacuoles), whereas relatively little moved into coated pits and vesicles. When studied using two-color laser confocal microscopy, internalized Texas Red-LPS and BODIPY-transferrin were found in different locations and failed to overlap completely even after extended incubation. In contrast, in THP-1 cells that expressed CD14 fused to the transmembrane and cytosolic domains of the low-density lipoprotein receptor, a much larger fraction of the cell-associated LPS moved into coated pits and colocalized with intracellular transferrin. These results suggest that CD14 (GPI)-dependent internalization of LPS occurs predominantly via noncoated plasma membrane invaginations that direct LPS into vesicles that are distinct from transferrin-containing early endosomes. A smaller fraction of the LPS enters via coated pits. Aggregation, which greatly increases LPS internalization, accelerates its entry into the nonclathrin-mediated pathway.

An NMR spectroscopy and molecular mechanics study of the molecular basis for the supramolecular structure of lipopolysaccharides.

Lipopolysaccharides from Gram-negative bacteria interact with the mammalian immune system to trigger a cascade of physiological events leading to a shock syndrome which results in the death in over 70% of cases of severe shock. It is known that the supramolecular structures of lipopolysaccharide aggregates are critical contributors to their biological activities. Despite this, the molecular basis for the formation if the regular hexagonal plates and arrays observed in lipopolysaccharide films and suspensions is unknown. Since these structures are two dimensional, it is unlikely that X-ray crystallographic methods will shed much light on their detailed structure. Knowing this structure is important since it is becoming increasingly likely that the insertion of the lipopolysaccharide hydrocarbon chains in the target host cell membrane may be involved in triggering host responses. This work describes the three-dimensional structure of the lipopolysaccharide lipid A moiety. The structure was obtained by a combination of molecular mechanics calculations and nuclear magnetic resonance spectroscopy. This involved calculation of the dihedral angle between the two glucosamine residues of the lipid A molecule from coupling constants and measuring critical interresidue NOE values. The study also takes into account information from X-ray powder diffraction and electron microscopy studies.

Crystallization of an R-form lipopolysaccharide from Klebsiella pneumoniae.

An R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (03-:K1-) formed crystals, whose shapes were elongated hexagonal plates, trapezoid plates, and rhomboid plates, and whose greatest dimensions were 3.1 x 0.8 microns, when it was suspended in 50 mM Tris buffer at pH 8.5 containing 5 mM MgCl2 and kept at 4 C for as long as 870 days. K. pneumoniae LEN-111 synthesized LPS molecules possessing incomplete repeating units of the O-antigenic polysaccharide portion besides the R-form LPS because of a leaky characteristic, but crystals consisted exclusively of the R-form LPS. Although the size of crystals was not large enough for X-ray analysis and limited crystallographic information was available, it was suggested that the crystals consist of hexagonal lattices with an alpha axis of 4.62 A and c axis of 79.8 +/- 2.6 A. The present results showed that R-form LPS lacking the O-antigenic polysaccharide portion tends to form crystals during long-term incubation in Tris buffer at pH 8.5 containing MgCl2 at 4 C.

Crystallization and analyses of crystals of various chemotypes of R-form lipopolysaccharides from Salmonella spp.

Various chemotypes (Re, Rd2, Rd1P-, Rd1, RcP-, Rc, Rb3, Rb2, Rb1, and Ra) of R-form lipopolysaccharides (LPSs) of Salmonella spp. were crystallized by treatment with 70% ethanol containing 250 mM MgCl2, and crystals of the LPSs were observed electron microscopically and analyzed by electron diffraction and synchrotron X-ray diffraction. All the LPSs tested formed three-dimensional crystals showing very similar shapes; hexagonal plate, solid column, discoid, square or rectangular plate, lozenge plate and truncated hexangular or rectangular pyramid forms. Electron diffraction patterns from the hexagonal plate crystals of all these LPSs obtained by electron irradiation from the direction perpendicular to the basal plane showed that they consist of hexagonal lattices with the lattice constant of 4.62 A. The crystals of all the LPSs thus formed gave ring-like X-ray diffraction patterns because of their small sizes. The long-axis values were calculated from the X-ray diffraction patterns from crystals of all the LPSs in the low-angle region and they corresponded roughly to the length of the proposed primary chemical structures of the R cores of the LPSs. The volume occupied by a single molecule of all the LPSs were calculated from the molecular weights based on the proposed structures and the crystallographic data obtained by electron diffraction, X-ray diffraction, and density determination.