Search

Cationic antimicrobial cationic peptides (CAMP) have been found in recent years to play a decisive role in hosts’ defense against microbial infection. They have also been investigated as a new therapeutic tool, necessary in particular due to the increasing resistance of microbiological populations to antibiotics. The structural basis of the activity of CAMPs has only partly been elucidated and may comprise quite different mechanism at the site of the bacterial cell membranes or in their cytoplasm. Polymyxin B (PMB) is a CAMP which is effective in particular against Gram-negative bacteria and has been well studied with the aim to understand its interaction with the outer membrane or isolated membrane components such as lipopolysaccharide (LPS) and to define the mechanism by which the peptides kill bacteria or neutralize LPS. Since PMB resistance of bacteria is a long-known phenomenon and is attributed to structural changes in the LPS moiety of the respective bacteria, we have performed a thermodynamic and biophysical analysis to get insights into the mechanisms of various LPS/PMB interactions in comparison to LPS from sensitive strains. In isothermal titration calorimetric (ITC) experiments considerable differences of PMB binding to sensitive and resistant LPS were found. For sensitive LPS the endothermic enthalpy change in the gel phase of the hydrocarbon chains converts into an exothermic reaction in the liquid crystalline phase. In contrast, for resistant LPS the binding enthalpy change remains endothermic in both phases. As infrared data show, these differences can be explained by steric changes in the headgroup region of the respective LPS.

Salmonella minnesota R 595 (Re) and other Salmonella strains incorporate cis-delta 9-16:1 (palmitoleic acid) at the expense of mainly dodecanoic acid into the lipid-A portion of lipopolysaccharides, when the cells are grown at low temperature (12 degrees C). It has recently been shown, that in S. minnesota R 595 grown at 37 degrees C dodecanoic acid is linked to the 3-hydroxyl group of an amide-bound 3-hydroxytetradecanoic acid. The present data suggest, that cis-delta 9-16:1 occupies the same position in lipid A (12 degrees C).

International audience; In this study, we aimed to evaluate the antioxidant and anti-inflammatory properties of Opuntia ficus-indica cactus cladode extracts in microglia BV-2 cells. Inflammation associated with microglia activation in neuronal injury can be achieved by LPS exposure. Using four different structurally and biologically well-characterized LPS serotypes, we revealed a structure-related differential effect of LPS on fatty acid β-oxidation and antioxidant enzymes in peroxisomes: Escherichia coli-LPS decreased ACOX1 activity while Salmonella minnesota-LPS reduced only catalase activity. Different cactus cladode extracts showed an antioxidant effect through microglial catalase activity activation and an anti-inflammatory effect by reducing nitric oxide (NO) LPS-dependent production. These results suggest that cactus extracts may possess a neuroprotective activity through the induction of peroxisomal antioxidant activity and the inhibition of NO production by activated microglial cells.

After about 70 years two new adjuvants have been approved for human vaccines. The first is MF59 developed by the ex-Chiron now Novartis Vaccines and it consists in an oil-in-water emulsion, comprising a low content of biodegradable squalene oil (4.3%) as the dispersed phase, which is stabilized by two non-ionic surfactants (Tween 80 and Span 85), and a low ionic strength citrate buffer as the continuous phase. The second one, defined as AS04, has been developed by GSK Biologics and consists in 3-0-desacyl-4'-monophosphoryl lipid A (MPL) that comes from the cell wall LPS of Gram-negative Salmonella minnesota R595 and is detoxified by mild hydrolytic treatment and purification. It is absorbed on aluminum hydroxide or aluminum phosphate. Thus, new molecules are available to improve the immune response to vaccines also in humans: this is the beginning of a new era in vaccinology.

Recently published studies showed that human hemoglobin (Hb) forms stable complexes with bacterial endotoxin (lipopolysaccharide, LPS), and that these complexes have much greater biological activity than LPS alone. LPS in LPS-Hb complexes has greater filterability, is less turbid, and has a lower sedimentation velocity compared to LPS alone, changes consistent with smaller LPS size. To further characterize the physical changes in LPS induced by Hb, we examined the ultrastructure of LPS alone and LPS-Hb complexes by negative staining. We studied two rough (Re) LPS and one smooth LPS, before and after incubation with purified human HbA0. Salmonella minnesota (Re) 595 LPS was visibly turbid, and was composed mostly of ribbon-like and mesh-like structures. In addition, this LPS contained large irregular membrane-like masses, and a few smaller discoidal particles. After incubation with Hb, none of the large mesh-like, ribbon-like or membrane-like structures remained; the resultant LPS particles were discoidal particles of 5-20 nm. Escherichia coli (Re) LPS alone was comprised predominantly of ribbon-like structures; after incubation with Hb, this LPS was comprised of circular membrane-like sheets (predominantly 40-200 nm). Smooth E. coli 055:B5 LPS alone contained heterogeneous particles of 10-100 nm. Most of the largest of these particles were disintegrated by the addition of Hb. The time course of Hb-induced LPS disaggregation was studied with S. minnesota LPS because the great turbidity of this LPS allowed us to monitor its disaggregation spectroscopically. Turbidity was decreased by 50% within approximately 5 h, and was not measurable by 25 h. We propose that the toxic domains of LPS (i.e. lipid A) are cryptic in native LPS aggregates and become exposed upon binding with Hb, thereby resulting in increased biological activity of LPS-Hb complexes compared to LPS alone.

The protein product of the early response gene, Egr-1, is required for macrophage differentiation. Egr-1 mRNA is induced by lipopolysaccharide within minutes in murine peritoneal macrophages through an effect on the rate of transcription of Egr-1. Therefore, we undertook a series of studies to characterize Egr-1 protein induced by LPS and to define enhancer elements in the Egr-1 promoter which mediate the effect of LPS in murine macrophages. Salmonella minnesota Re 595 LPS (10 ng/ml or higher) induced Egr-1 protein of 80 kDa within 45 min in thioglycollate-elicited peritoneal macrophages. The promoter region of Egr-1 contains six serum response elements (SRE) which each contain a core CArG box. CAT activity in LPStreated macrophages transfected with Egr-1-CAT constructs containing the two most upstream 5' SREs was increased 4.7-fold whereas CAT activity in macrophages transfected with constructs lacking these two most upstream SREs was not affected by LPS. LPS induced CAT activity in macrophages transfected with constructs containing tetramers of each of the two most 5' SREs upstream of a heterologous promoter linked to CAT. Mutation of the CArG box abolished the effect of LPS. These findings suggest that the CArG box elements within the two most upstream SREs may mediate the effect of LPS on transcription of Egr-1.

The phenol-chloroform-petroleum ether (PCP) extraction is the method of choice for isolating LPS chemotypes from rough mutant bacteria. However, we have observed that a high percentage of PCP-purified LPS preparations contain trace levels of protein contaminants, and these protein contaminants may exhibit endotoxin-like activity. To obtain protein-free rough LPS, a modified phenol-water extraction procedure was developed for use as a final step to follow PCP extraction. Using this procedure, Salmonella minnesota Ra, Rc, Rd and Re LPS were recovered in yields of 82-100% from the original PCP-extracted preparations. Yields were determined by recovery of KDO and supported by analysis of silver stained SDS-PAGE gels. Although the original PCP-purified chemotypes were contaminated by major 41, 38.5, 29.5, 23, 17 and 14 kDa proteins (detected by Western blotting and gold staining), the repurified LPS contained no detectable protein. The repurified LPS retained all of its original bioactivity as defined by the ability to stimulate normal C3H/OuJ macrophages to secrete TNF. The importance of utilizing repurified LPS in biologic studies was illustrated by the loss of > 99% of activity when repurified LPS was used to stimulate TNF secretion by LPS 'hyporesponsive' C3H/HeJ macrophages.

The patterns of susceptibility to hydrophobic and hydrophilic drugs and the uptake of the fluorescent probe N-phenyl-naphthylamine in Brucella spp., Haemophilus influenzae, Escherichia coli, and deep rough Salmonella minnesota mutants were compared. The results show that the outer membranes of smooth and naturally rough Brucella spp. do not represent barriers to hydrophobic permeants and that this absence of a barrier relates at least in part to the properties of Brucella lipopolysaccharide.

This Midterm report details new analytical approaches developed for the determination of endotoxin structures at much improved sensitivity and specificity, and an application of those techniques to Coxiella burnetti LPS. Foremost in this methodology is a reducing-end reagent synthesized to provide femtomole (10-15) detection for 0-antigen oligosaccharides. A second development focuses on methodology to extract the details of linkage, branching, and sequence, by periodate oxidation, supercritical fluid chromatography, and mass spectrometry. These techniques combine to provide the elements of a global approach to oligosaccharide structure. The utility of supercritical fluid chromatography for a determination of Lipid-A from Salmonella minnesota has been demonstrated. Finally, the structural features of Lipid-A from Coxiella burnetii is described using a combination of these newly developed analytical techniques. Keywords: Lipopolysaccharide; Immunogen; Coxiella burnetii.

It is not clear whether baboons develop fever in response to endotoxin or other pyrogens. We injected various pyrogens intravenously in 12 unrestrained baboons (Papio ursinus) and measured their body temperature using intra-abdominal radio-telemeters. Serum iron concentration was also measured. The baboons developed fever after injection of killed Staphylococcus aureus (5 × 107 organisms/kg). No significant fever was measured after injection of lipopolysaccharide (Salmonella typhosa) (0.1, 8, 40, and 100 μg/kg), bovine serum albumin (4 mg/kg), killed Salmonella minnesota (5 × 107 organisms/kg), and killed Salmonella typhi (5 × 107 organisms/kg). A significant decrease in serum iron concentration was found only after injection of S. aureus and lipopolysaccharide, 100 μg/kg. The phagocytic synthesis of interleukin-1 following pyrogen stimulation in baboons and some other primates appears to differ from that in man and in nonprimates.

An IgG1 monoclonal antibody, Sulph I, reacting with sulphatide (3′-sulphogalactosylceramide), was produced by immunizing Balb/c mice with that glycolipid coated on Salmonella minnesota bacterial membrane. Radioimmunodetection of the binding of the monoclonal antibody to structurally related glycolipids adsorbed to microtitre plates or chromatographed on thin-layer plates was used to determine its binding epitope. The antibody showed similar binding avidity to three sulphated glycolipids: sulphatide, sulpholactosylceramide and seminolipid. Lysosulphatide did bind the antibody, but, compared with sulphatide, 30 times more antigen was needed for half-maximal binding. Bis(sulphogangliotriosyl)ceramide and bis-sulphogangliotetraosylceramide did not bind the antibody. These results suggest that terminal galactose-3-O-sulphate and part of the hydrophobic region of the glycolipid are recognized by the Sulph I antibody.

Smooth strains of Salmonella typhimurium and S. minnesota, and chemotypes Ra, Rb, and Rc, which are deficient in lipopolysaccharide components of the somatic side chains and outer core region, grow normally on nutrient agar and nutrient broth up to 45 degrees C. However, most mutants with defects in the heptose region of the LPS (chemotypes Rd2 and Re) do not grow on this medium at 42 degrees C or above; a few grow at 42 degrees C but not at 45 degrees C. In liquid medium (nutrient broth, or phosphate minimal medium), growth, measured as turbidity or as colony-forming units, stops 60 to 90 min after shift from 30 to 42 degrees C; DNA and protein synthesis cease at the same time. Growth does not reoccur at 42 degrees C; protein synthesis and growth reinitiate upon shift to 30 or 37 degrees C. Growth cessation does not alter cell morphology in the phase-contrast microscope. Growth of heptose-deficient strains at 42 degrees C in nutrient broth is restored by MgCl2 (0.5 mM), NaCl (50 mM), or sucrose (100 mM). Sensitivity to smooth-specific and rough-specific phages, and analysis of LPS composition, indicate that heptose-deficient mutants grown at temperatures from 30 to 45 degrees C, and in the presence or absence of high salt, do not contain heptose or O-specific sugars in their LPS.

Benzyl-3- O -benzyl-2-benzyloxycarbonylamino-6- O -[2-benzyloxycarbonyl-amino-2-deoxy-3,4- O -(tetraisopropyldisiloxane-1,3-diyl)- β- d -glucopyranosyl]-2-deoxy-α- d -glucopyranoside was coupled with methyl (4,5,7,8-tetra- O -acetyl-3-deoxy-α- d - manno -2-octulopyranosyl bromide)onate ( 13 ) to yield the α-glycosidically linked trisaccharide. After deacetylation and selective introduction of a second 7′,8′- O -tetraisopropyldisiloxane group, a further glycosidation reaction with 13 led regioselectively to the tetrasaccharide benzyl O -[methyl (4,5,7,8-tetra- O -acetyl-3-deoxy-α- d - manno -2-octulopyranosyl)onate]-(2→4)- O -{methyl [3-deoxy-7,8- O -(tetraisopropyldisiloxane-1,3-diyl)-α- d - manno -2-octulopyranosyl]-onate}-(2→6)- O - [2-benzyloxycarbonylamino-2-deoxy-3,4- O -(tetraisopropyldisiloxane-1,3-diyl)-β- d -glucopyranosyl]- (1→6)-3- O -benzyl-2-benzyloxycarbonyl-amino-2-deoxy-α- d -glucopyranoside. A series of deblocking steps gave O -(3-deoxy-α- d - manno -2-octulopyranosylonic acid)-(2→4)- O -(3-deoxy-α- d - manno -2-octulopyranosylonic acid)- (2→6)- O -(2-amino-2-deoxy-β- d -glucopyranosyl)-(1→6)-2-amino-2-deoxy- d -glucopyranose which was identical with a tetrasaccharide that had been isolated by hydrazinolysis of the lipopolysaccharide from Salmonella minnesota R 595. Hence, synthetic proof is provided for the linkages in this part of the inner core region of lipopolysaccharides.

An enzyme-linked immunosorbent assay was developed which allowed the measurement of rabbit immunoglobulin G antibodies directed to Salmonella minnesota Re glycolipid. Efficient adsorption of the antigen to polystyrene could only be effected provided it had been previously dialyzed against 0.2 M EDTA (pH 7.0) and subsequently treated with 0.2% sodium deoxycholate (15 min at 56 degrees C) in 0.05 M diethanolamine buffer (pH 9.6). The method is by far more sensitive than quantitative precipitation in the determination of IgG antibodies. Inhibition by glycolipid of binding S. minnesota Re antibodies to immobilized glycolipid attests to the specificity of the assay and permits the detection of as little as 100 ng of glycolipid.

The role of the lipopolysaccharide (LPS) structure in the recipient cell during bacterial conjugation was studied using a series of well-defined LPS mutations in Salmonella minnesota. The plasmids Flac (IncFI) and R1drd19 (IncFII) transferred at a high frequency to the smooth S218 parent strain and the rough LPS mutants. However, R64drd11 (IncIα) transferred poorly to the LPS mutants compared with transfer to the smooth LPS parent strain. The decrease in R64drd11 transfer frequency correlated with the extent of the defect in LPS structure, suggesting that intact LPS on the recipient is a major requirement for R64drd11 mating. Transfer of the P-group plasmid, RK2, was not affected by changes in LPS structure. These results show that plasmids use different cell surface structures during conjugation, and that LPS is particularly important for R64drd11 transfer.

SUMMARY: Isolated lipopolysaccharide from a rough mutant of Salmonella minnesota was shown to act as a receptor for Felix 0-1 bacteriophage. The isolated receptor structure could be dissociated by sodium deoxycholate with subsequent loss of receptor activity. When the deoxycholate was removed by dialysis the receptor activity was restored. Ultrasonic treatment destroyed the receptor activity and electron micrographs indicated that the ultrasonic treatment had altered the structure of the lipopolysaccharide. The experiments suggest that the inactivation of phages can be used as a test of the biological activity of a lipopolysaccharide. The interaction between the 0-1 phage and the receptor structure was also studied with the aid of electron microscopy.

The nature of the cell surface constituent(s) containing sialic acid that block fixation of bacterial glycolipid mR595 to normal rat fibroblasts was explored. Indirect evidence suggests that a trypsin-sensitive, high molecular weight glycoprotein, present in larger amounts on the surface of normal cells (as compared with transformed rat fibroblasts), may be involved as a blocking agent.

S-form lipopolysaccharides (LPS) from Klebsiella strain LEN-1 (O3: K1 —) and from Salmonella minnesota strain 1114 were positively stained with ruthenium red, whereas R-form LPS from Klebsiella strain LEN-111 (O3— : K1 —) and Ra, Rb1, RcP+, Rd1P-, and Re LPS from the respective mutant strains of S. minnesota were not or only faintly stained by such treatment. From these results it was concluded that ruthenium red stains the O-specific polysaccharide chains of LPS. The appearance of stained preparations of S-form LPS suggested that the material responsible for this positive staining corresponded to the surface projections which were seen by the negative staining technique as attached to the ribbon-like structures and spherules of the LPS.

Lipopolysaccharides (LPS) are well known inducers of interleukin 1 (IL 1). Here we show that synthetic heptaacyl Salmonella minnesota (compound 516) and synthetic E. coli type (compound 506) lipid A, as well as monodephospho part structures thereof, are able to induce IL 1 production in human mononuclear cells. The 4'-monodephospho part structure (compound 505) was found to be the most active IL 1 inducer as compared to compound 506 and the 1-monodephospho part structure (compound 504). Synthetic lipid A precursor Ia, lacking nonhydroxylated fatty acids (compound 406), and its 1- or 4'-monodephospho part structures (compounds 404 and 405) did not induce IL 1 production. IL 2 is not produced during stimulation with the synthetic compounds. IL 1 activity was partially heat labile and could be inhibited by a rabbit antiserum against human leukocytic pyrogen. These results show that the lipid A component of LPS is active in inducing IL 1 and that the presence of 3-acyloxyacyl residues is necessary for IL 1 induction.

Normal rabbit serum reduces the buoyant density of lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (d = 1.44 g/cm3) and Salmonella minnesota R595 (d = 1.38 g/cm3) to a value less than g/cm3. This density shift is associated with the inhibition of a number of endotoxic activities of the LPS; namely, the pyrogenic activity, the ability to produce an immediate neutropenia in rabbits, lethality in adrenalectomized mice, and anticomplementary activity. A qualitatively similar change in buoyant density was observed to occur after intravenous injection of the LPS into rabbits. Preliminary evidence suggests that the density shift does not occur as a result of the degradation of the glycolipid backbone of the LPS. These data suggest that the interactions of LPS with plasma (or serum) components leading to reduction in buoyant density may account for a major pathway of LPS detoxification.

SUMMARY: Envelope proteins of one smooth (S) strain and seven rough (R) mutants of Salmonella minnesota were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. All strains gave similar band patterns although some consistent differences were detected. A major polypeptide band at 54k, which coincided with the flagellar component, was more prominent in S, Ra and Rb than in the Rc, Rd and Re chemotypes. The latter strains, however, showed more prominent bands at 48, 19 and 18k. The stage of growth at which the cultures were harvested was also found to affect the band patterns, particularly in the 54 and 40k regions. A closer examination of S, Ra and Re strains suggested that the levels of the major 40 and 37k bands were slightly reduced in Re. It is concluded that the progressive loss of lipopolysaccharide components which occurs from the S chemo-type through various degrees of roughness to Re is accompanied by a change in the envelope protein composition, apparently between Rb and Rc.

From the lipid A backbone of Salmonella lipopolysaccharide, β-1,6-linked glucosamine disaccharides had been isolated previously, which were substituted at the non-reducing glucosamine unit with phosphate and 2-keto-3-deoxyoctonate residues. The results now obtained by periodate degradation show that the phosphate group is linked to the 4 position and the 2-keto-3-deoxy-octonate oligosaccharide probably to the 3 position of the non-reducing glucosamine unit.