Bacteroides Lipopolysaccharides Research Articles

A possible beneficial effect of Bacteroides on faecal lipopolysaccharide activity and cardiovascular diseases

Faecal lipopolysaccharides (LPS) have attracted attention as potent elements to explain a correlation between the gut microbiota and cardiovascular disease (CVD) progression. However, the underlying mechanism of how specific gut bacteria contribute to faecal LPS levels remains unclear. We retrospectively analysed the data of 92 patients and found that the abundance of the genus Bacteroides was significantly and negatively correlated with faecal LPS levels. The controls showed a higher abundance of Bacteroides than that in the patients with CVD. The endotoxin units of the Bacteroides LPS, as determined by the limulus amoebocyte lysate (LAL) tests, were drastically lower than those of the Escherichia coli LPS; similarly, the Bacteroides LPS induced relatively low levels of pro-inflammatory cytokine production and did not induce sepsis in mice. Fermenting patient faecal samples in a single-batch fermentation system with Bacteroides probiotics led to a significant increase in the Bacteroides abundance, suggesting that the human gut microbiota could be manipulated toward decreasing the faecal LPS levels. In the clinical perspective, Bacteroides decrease faecal LPS levels because of their reduced LAL activity; therefore, increasing Bacteroides abundance might serve as a novel therapeutic approach to prevent CVD via reducing faecal LPS levels and suppressing immune responses.

Variation in Microbiome LPS Immunogenicity Contributes to Autoimmunity in Humans

According to the hygiene hypothesis, the increasing incidence of autoimmune diseases in western countries may be explained by changes in early microbial exposure, leading to altered immune maturation. We followed gut microbiome development from birth until age three in 222 infants in Northern Europe, where early-onset autoimmune diseases are common in Finland and Estonia but are less prevalent in Russia. We found that Bacteroides species are lowly abundant in Russians but dominate in Finnish and Estonian infants. Therefore, their lipopolysaccharide (LPS) exposures arose primarily from Bacteroides rather than from Escherichia coli, which is a potent innate immune activator. We show that Bacteroides LPS is structurally distinct from E.coli LPS and inhibits innate immune signaling and endotoxin tolerance; furthermore, unlike LPS from E.coli, B.dorei LPS does not decrease incidence of autoimmune diabetes in non-obese diabetic mice. Early colonization by immunologically silencing microbiota may thus preclude aspects of immune education.

Biological activity of Bacteroides lipopolysaccharide--reappraisal.

Lipopolysaccharide (LPS) was extracted from six species of Bacteroides by the phenol/water, petroleum/chloroform/ phenol, and Triton/magnesium methods. Yields and chemical analysis demonstrated that the products were different. Biological activity (endotoxicity) was assessed by the limulus amebocyte lysate (LAL) assay, induction of tumor necrosis factor (TNF) from human mononuclear leukocytes, and a lethality model with galactosamine-sensitized mice. Results showed that endotoxicity varied greatly depending on the species and the extraction method. LPS prepared by the phenol/water method was most endotoxic and that from Bacteroides fragilis had the greatest activity. Compared with Escherichia coli LPS, the phenol/water extract of B. fragilis was sevenfold more active in the LAL assay and marginally less active (five-to seven-fold) in the bioassay for TNF induction. However, when B. fragilis LPS was added to E. coli LPS, the induction of TNF was inhibited. In the mouse model, B. fragilis LPS was 5,000-fold less toxic. If the gastrointestinal tract is the source of the endotoxin in patients with systemic inflammatory response syndrome, then the obligately anaerobic Bacteroides species, which outnumber the facultative species such as E. coli by 1,000-fold, should not be overlooked.

Tumor necrosis factor induction by an aqueous phenol-extracted lipopolysaccharide complex from Bacteroides species

The stimulation of macrophages and monocytes by lipopolysaccharide (LPS) results in the secretion of tumor necrosis factor (TNF), a cytokine which is thought to play a pivotal role in subsequent host responses. Its induction is thought to be facilitated by the binding of complexes of LPS and LPS-binding protein to CD14. The LPS of Bacteroides species was considered a weak endotoxin; however, in a recent study we have shown that the biological activity and chemical composition of the LPS from Bacteroides species are dependent on the extraction method. The present study determines the capacity of LPS extracted by aqueous phenol (the method for producing an LPS of high endotoxic activity) from four species of Bacteroides to induce TNF. Induction was investigated from human mononuclear leukocytes (MNL), THP-1 cells (with and without enhancement by vitamin D2 for CD14), and peritoneal macrophages from C3H/HeJ (LPS nonresponder) and C3H/HeN (LPS responder) mice. Escherichia coli O18K- LPS, a typical smooth LPS of heterogeneous molecular mass, was used as a control throughout. The stimulation of TNF production by E. coli LPS was between two- and fourfold more than that by Bacteroides LPS in MNL, in THP-1 cells (with enhancement for CD14), and in peritoneal macrophages from C3H/HeN mice. In THP-1 cells (without enhancement for CD14), there was no significant difference in TNF production between E. coli and Bacteroides LPSs. In peritoneal macrophages from C3H/HeJ mice, E. coli LPS stimulated no TNF production, but there was no significant difference in TNF production from peritoneal macrophages from C3H/HeJ and C3H/HeN mice by Bacteroides LPS. In all cell populations, there was a peak of TNF production after approximately 4 h of stimulation with all LPSs tested. However, other peaks of TNF production were seen in MNL and THP-1 cells (with enhancement for CD14) after stimulation with E. coli LPS only. In stimulation assays in which Bacteroides LPS was together with but in excess of E. coli LPS, it was found that TNF production from MNL and THP-1 cells (with and without enhancement for CD14) was comparable to that of Bacteroides LPS alone and not E. coli LPS alone. An anti-CD14 monoclonal antibody did not inhibit Bacteroides LPS-stimulated TNF production. However, E. coli LPS-stimulated TNF release was inhibited by an anti-CD14 monoclonal antibody, most noticeably in MNL and THP-1 cells (with enhancement for CD14).(ABSTRACT TRUNCATED AT 400 WORDS)

A re-appraisal of the biological activity of bacteroides LPS

Lipopolysaccharides (LPS) were extracted from seven Bacteroides strains by three different techniques: the phenol-water (PW), phenol-chloroform-petroleum (PCP) and Triton-Mg2+ methods. The strains selected included two different B. fragilis strains, one of which was grown in two different media. Yields varied between the strains, growth media and extraction technique, but generally the highest yield by weight was from the PCP method and the lowest from the PW method. The PW method was selected for the greatest amounts of carbohydrate and KDO, and the PCP method for the least. Phosphorus levels were more uniform among all extraction methods. Protein contamination was found in all Bacteroides LPS extracts, with extremely low levels in PW-LPS and the highest levels in material extracted by the PCP and Triton-Mg2+ techniques. No protein contamination could be detected after proteinase K treatment. After silver staining LPS PAGE profiles showed ladder patterns characteristics of smooth LPS for B. vulgatus, B. thetaiotaomicron and the control Escherichia coli O18:K- strains, whereas the other Bacteroides strains showed mainly rough and low M(r) material only. The PCP method did not select for high M(r) material in the B. fragilis strains; otherwise the LPS profiles for all extraction methods were identical. The biological activities of native and sodium salt form LPS were investigated on a weight for weight basis and compared to that of E. coli O18:K- PW-LPS. Amongst the LPS from Bacteroides strains, those prepared by the PW method were found to have a significantly higher activity in a galactosamine mouse lethality model, in induction of TNF and the Limulus amoebocyte lysate (LAL) assay, than LPS extracted by the PCP or Triton-Mg2+ methods. LPS from Bacteroides strains extracted by the PCP method had consistently low activity in all assays. Comparing PW-LPS from Bacteroides strains with that from E. coli O18:K- in the galactosamine mouse model, the E. coli O18:K- LPS was c. 5000-fold more active than the most active bacteroides LPS. However, in the LAL assay native PW-LPS from both the B. fragilis strains, and B. caccae had higher activities (up to 30-fold) than E. coli O18:K- LPS, with the PW-LPS from the other Bacteroides spp. being up to 15-fold less active than the E. coli O18:K- PW-LPS. In the TNF induction assay, E. coli O18:K- PW-LPS was 4-50-fold more active than bacteroides PW-LPS.(ABSTRACT TRUNCATED AT 400 WORDS)

The resolution of bacteroides lipopolysaccharides by polyacrylamide gel electrophoresis

The lipopolysaccharides (LPS) of the 10 species of the genus Bacteroides (sensu stricto) were extracted by the proteinase K method and their resolution compared by several methods of polyacrylamide gel electrophoresis (PAGE). These included sodium dodecyl sulphate (SDS)-PAGE with and without urea, polyacrylamide gradient gels and Tricine [N-Tris (hydroxymethyl) methyl glycine]-SDS-PAGE. The original discontinuous system showed good resolution of LPS from B. thetaiotaomicron, B. caccae and B. ovatus and this was enhanced by urea; B. vulgatus showed a typical ladder pattern associated with repeating polysaccharide units of the O side chains. The LPS profiles of the other species, including B. fragilis, were poorly resolved; the majority of components migrated with the leading edge of the wave front. The resolution of the LPS of these species was marginally improved with gradient gels but the majority of components were separated only within the regions of high polyacrylamide concentration. The Tricine-SDS system was consistently superior to the other methods, with excellent resolution of the LPS profiles of all Bacteroides species.

Induction of interleukin-1 and -6 in human gingival fibroblast cultures stimulated with Bacteroides lipopolysaccharides

Normal human gingival fibroblasts stimulated in vitro by lipopolysaccharides (LPS) from oral Bacteroides species produced cell-free and cell-associated thymocyte-activating factors (TAF). Neutralization assays using antisera to human interleukin-1 alpha (HuIL-1 alpha), HuIL-1 beta, and HuIL-6 revealed that cell-free TAF was attributable mainly to IL-1 beta and that IL-6 augmented the TAF activity of IL-1 beta in the culture supernatant. Another factor(s), however, may also be involved in cell-free TAF. By contrast, the active entity of cell-associated TAF was ascribed to IL-1 alpha alone. Furthermore, IL-6 was detected mainly in the supernatant of fibroblast cultures stimulated with Bacteroides LPS. Fibroblasts pretreated with natural human beta or gamma interferon, but not those pretreated with alpha interferon, synthesized higher levels of cell-associated IL-1 alpha in response to stimulation by Bacteroides LPS; however, no interferons exhibited direct IL-1-inducing activity or synergistic IL-1-inducing activity with LPS. Endogenously induced beta interferon was suggested to be necessary for fibroblasts to produce cell-associated IL-1 alpha in response to Bacteroides LPS.

Bacteroides Lipopolysaccharides (LPS) Induce Anaphylactoid and Lethal Reactions in LPS-Responsive and -Nonresponsive Mice Primed with Muramyl Dipeptide

Lipopolysaccharides (LPS) (100 micrograms/mouse) from Bacteroides gingivalis elicited anaphylactoid reactions in N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) (100 micrograms)-primed C3H/HeN mice 0-48 h after MDP injection. The reaction resulted in death within 1 h when the LPS was injected 2-10 h after the MDP injection. LPS prepared from other Bacteroides and Salmonella species but not from others, including Escherichia coli, could induce the reaction. LPS-nonresponsive C3H/HeJ and C57BL/6 and C57BL/10 strains were also responsive to the reaction, while AKR, BALB/c, DBA/2, and ICR mice were not. By contrast, Bacteroides LPS exhibited weak lethal toxicity compared with E. coli LPS in galactosamine-sensitized C3H/HeN mice, while neither LPS had activity in C3H/HeJ mice. LPS from E. coli and Bacteroides intermedius induced serum tumor necrosis factor (TNF) activity in MDP-primed mice, except for C3H/HeJ mice; B. gingivalis LPS rarely induced TNF in C3H/HeN or C3H/HeJ mice, indicating no involvement of TNF in the anaphylactoid reaction. Serotonin could substitute for MDP for the reaction, and methysergide, a serotonin antagonist, inhibited the activity.

Characterization and immunobiologic activities of lipopolysaccharides from periodontal bacteria.

Bacterial surface structures play a critical role in the initiation of infectious diseases. Various surface components of pathogenic bacteria have been reported to be involved in host injury. There is a great deal of evidence incriminating certain Gram-negative, anaerobic bacteria present in the gingival crevice as etiologic agents of human periodontal diseases. We have isolated endotoxic cellular components from suspected periodontopathic bacteria and examined their immunobiological activities. Lipopolysaccharides (LPS) and lipid-associated proteoglycans (LPG) were prepared from whole cells by the phenol-water and butanol-water procedures, respectively. LPS from Bacteroides gingivalis, B. intermedius, B. oralis, and B. loescheii, Fusobacterium nucleatum and F. necrophorum, and Actinobacillus (Haemophilus) actinomycetemcomitans were found to possess biological activities comparable with those of LPS from E. coli K235 in terms of activation of Limulus lysate, B-cell mitogenicity, polyclonal B-cell activation, induction of bone resorption, and IL-1 production by macrophages. These LPS contained mainly sugars, amino sugars, and fatty acids. No heptose or 2-keto-3-deoxyoctonate (KDO) was detected in the Bacteroides LPS, while LPS from Actinobacillus and Fusobacterium species contained significant amounts of heptose as well as small quantities of KDO. Bacteroides LPS were clearly mitogenic for spleen cells of C3H/HeJ mice, which are non-responsive to LPS from E. coli, A. actinomycetemcomitans, and Fusobacterium species. Furthermore, polymyxin B was found to abrogate the mitogenic activity of LPS from E. coli, Actinobacillus, and Fusobacterium species, but not those from Bacteroides species. Spleen cells from both C3H/HeN and C3H/HeJ mice responded to all butanol-water-extracted LPG preparations, including those from E. coli, A. actinomycetemcomitans, and Fusobacterium species. It may be concluded that LPS and LPG isolated from suspected periodontopathic bacteria possess marked immunobiological potencies on lymphoreticular and bone cells.

Gamma interferon modulation of prostaglandin E2 release from monocytes stimulated with lipopolysaccharides from Bacteroides intermedius, Bacteroides gingivalis, and Salmonella typhimurium.

Lipopolysaccharides (LPS) from Bacteroides species are less potent than enterobacterial LPS preparations when compared by pyrogenicity and bone resorption assays and by the localized Schwartzman reaction. In this investigation, LPS preparations from Bacteroides intermedius and Bacteroides gingivalis were examined for their capacity to stimulate PGE2 release from human monocytes relative to identical doses of Salmonella typhimurium LPS. In addition, the capacity of recombinant gamma interferon (IFN‐γ) to potentiate PGE2 release was examined with each LPS preparation. Monocytes, separated by counter flow centrifugation, were exposed to LPS (1 ng/ml, 100 ng/ml and 10 ng/ml) alone or in combination with IFN‐γ (10 Units/ml). PGE2 levels (measured by RIA) were determined in monocyte culture supernatants exposed during the following incubation periods: 0‐8, 8‐24, 24‐48, and 48‐72 h. Monocytes treated with Salmonella LPS released 2 to 3‐fold greater amounts of PGE2 than cells treated with Bacteroides species LPS preparations. Although IFN‐y alone did not stimulate PGE2 release, Bacteroides‐elicited PGE2 release was siginificantly increased when monocytes were cultured concomitantly with IFN‐γ. PGE2 release elicited by Salmonella LPS was not significantly affected by interferon. These findings indicate that IFN‐γ can markedly potentiate the release of PGE2 from monocytes stimulated with Bacteroides LPS. Furthermore, the observed weakness of these LPS preparations relative to Salmonella LPS can be largely overcome by co‐treating monocytes with IFN‐γ.

The capacity of lipopolysaccharides from bacteroides, fusobacterium and veillonella to produce skin inflammation and the local and generalized Shwartzman reaction in rabbits.

Purified lipopolysaccharides (LPS) from strains of Bacteroides, Fusobacterium and Veillonella were tested for endotoxic activity, utilizing the primary skin reaction and the Shwartzman phenomena in rabbits. Comparisons were made to Salmonella LPS. Each LPS preparation produced the primary skin reaction in a typical dose‐response fashion. Skin sites injected with 400 μg of Fusobacterium LPS often produced necrotic lesions. In the comparisons made between the skin lesion doses (SLD50) of the Veitlonella LPS and the Salmonella LPS, the difference was either non‐significant or the Salmonella LPS was only slightly more active (P <0.02). LPS from the Fusobacterium and the Bacteroides were significantly less toxic than the Salmonella LPS (P <0.005). All LPS preparations did prepare the skin for the local and the specific organs for the generalized Shwartzman reaction. The toxicity of the Bacteroides LPS was, however, very low in the local reaction (P <0.005). For all LPS preparations the sensitivity of the primary skin test (SLD50) was superior to the local Schwartzman reaction (SPD50) (P <0.005).Twenty‐four hours after challenge for the generalized Shwartzman reaction, extensive fibrin thrombi in the capillaries of the renal glomeruli were found, as well as thrombi containing necrotic blood cells, both in the veins of the liver and in the arteries of the lungs.The high endotoxic activity of the Fusobacterium and the Veillonella LPS may reflect a similarity between their lipid A and that of the Salmonella LPS.

Lethality for mice and chick embryos, pyrogenicity in rabbits and ability to gelate lysate from amoebocytes of Limulus polyphemus by lipopolysaccharides from Bacteroides, Fusobacterium and Veillonella.

Phenol-water extracted lipopolysaccharides (LPS) from Veillonella, Fusobacterium nucleatum, Bacteroides fragilis and Bacteroides melaninogenicus were lethal for mice and 11-days-old chick embryos, pyrogenic in rabbits, and gelated Limulus amoebocyte lysate. Mouse lethality was considerably enhanced by actinomycin-D. In all test systems the endotoxin activity of Veillonella and Fusocbacterium LPS was comparable to that of LPS from Salmonella enteritidis, which was included as a reference endotoxin. The endotoxicity of the Bacteroides LPS was very low. While nanograms of the Veillonella and Fusobacterium LPS killed the chick embryos and gelated the Limulus lysates, microgram amounts of the Bacteroides LPS were needed to give positive reaction in the same test systems. As much as 74 microgram of the most active B. fragilis LPS were required to give a typical biphasic fever response in rabbits. A significant correlation was found between all test results (r = 0.90-0.98, p less than 0.001).