ABSTRACT
The site-selective functionalization of carbohydrates is an active area of research. Reported here is the surprising observation that the sterically encumbered adamantyl group directed site-selective acylation at the C2 position of S-glycosides through dispersion interactions between the adamantyl C-H bonds and the πâ system of the cationic acylated catalyst, which may have broad implications in many other chemical reactions. Because of their stability, chemical orthogonality, and ease of activation for glycosylation, the site-selective acylation of S-glycosides streamlines oligosaccharide synthesis and will have wide applications in complex carbohydrate synthesis.
Subject(s)
Acylation/immunology , Oligosaccharides/chemistry , Catalysis , Glycosylation , HumansABSTRACT
Salmonella enterica serovar Typhimurium (S. Typhimurium) changes the structure of its lipopolysaccharide (LPS) in response to the environment. The two main LPS variants found in S. Typhimurium correspond to LPS with a hepta-acylated lipid A (LPS 430) and LPS with modified phosphate groups on its lipid A (LPS 435). We have previously shown that these modified LPS have a lower capacity than wild type (WT) LPS to induce the production of pro-inflammatory cytokines in mice. Nevertheless, it is not know if LPS 430 and LPS 435 could also subvert the innate immune responses in human cells. In this study, we found that LPS 430 and LPS 435 were less efficient than WT LPS to induce the production of pro-inflammatory cytokines by human monocytes, in addition we found a decreased dimerization of the TLR4/MD-2 complex in response to LPS 430, suggesting that structurally modified LPS are sensed differently than WT LPS by this receptor; however, LPS 430 and 435 induced similar activation of the transcription factors NF-κB p65, IRF3, p38 and ERK1/2 than WT LPS. Microarray analysis of LPS 430- and LPS 435-activated monocytes revealed a gene transcription profile with differences only in the expression levels of microRNA genes compared to the profile induced by WT LPS, suggesting that the lipid A modifications present in LPS 430 and LPS 435 have a moderate effect on the activation of the human TLR4/MD-2 complex. Our results are relevant to understand LPS modulation of immune responses and this knowledge could be useful for the development of novel adjuvants and immunomodulators.
Subject(s)
Cytokines/immunology , Inflammation/immunology , Lipopolysaccharides/immunology , Lymphocyte Antigen 96/immunology , Monocytes/immunology , Salmonella typhimurium/immunology , Toll-Like Receptor 4/immunology , Acylation/immunology , Dimerization , Humans , Inflammation/microbiology , Lipid A/immunology , Monocytes/microbiology , Salmonella Infections/immunology , Salmonella Infections/microbiology , Signal Transduction/immunology , Transcription Factors/immunology , Transcription, Genetic/immunologyABSTRACT
Shigellosis, a major cause of diarrhea worldwide, exhibits high morbidity and mortality in children. Specificity of Shigella immunity is determined by the structure of the main protective O-antigen polysaccharide component incorporated into the lipopolysaccharide (LPS) molecule. Endotoxicity, however, precludes LPS clinical use. Thus, there is still no vaccine against the most prevalent shigellosis species (serotype S. flexneri 2a), despite ongoing efforts focused on inducing serotype-specific immunity. As LPS is highly heterogenous, we hypothesized that more homogenous pools of LPS might be less toxic. We developed a method to generate a homogenous S. flexneri 2a LPS subfraction, Ac3-S-LPS, containing long chain O-specific polysaccharide (S-LPS) and mainly tri-acylated lipid A, with no penta- and hexa-acylated, and rare tetra-acylated lipid A. Ac3-S-LPS had dramatically reduced pyrogenicity and protected guinea pigs from shigellosis. In volunteers, 50⯵g of injected Ac3-S-LPS vaccine was safe, with low pyrogenicity, no severe and few minor adverse events, and did not induce pro-inflammatory cytokines. In spite of the profound lipid A modification, the vaccine induced a prevalence of IgG and IgA antibodies. Thus, we have developed the first safe immunogenic LPS-based vaccine candidate for human administration. Homogenous underacetylated LPSs may also be useful for treating other LPS-driven human diseases. Clinical trial registry: http://grls.rosminzdrav.ru/.
Subject(s)
Acylation/immunology , Dysentery, Bacillary/immunology , Lipopolysaccharides/immunology , Shigella Vaccines/immunology , Shigella flexneri/immunology , Animals , Antibodies, Bacterial/immunology , Bacterial Proteins/immunology , Cell Line, Tumor , Guinea Pigs , Humans , O Antigens/immunology , U937 CellsABSTRACT
O-linked ß-N-acetyl glucosamine modification (O-GlcNAcylation) is a dynamic, reversible posttranslational modification of cytoplasmic and nuclear proteins. O-GlcNAcylation depends on nutrient availability and the hexosamine biosynthetic pathway (HBP), which produces the donor substrate UDP-GlcNAc. O-GlcNAcylation is mediated by a single enzyme, O-GlcNAc transferase (OGT), which adds GlcNAc and another enzyme, O-GlcNAcase (OGA), which removes O-GlcNAc from proteins. O-GlcNAcylation controls vital cellular processes including transcription, translation, the cell cycle, metabolism, and cellular stress. Aberrant O-GlcNAcylation has been implicated in various pathologies including Alzheimer's disease, diabetes, obesity, and cancer. Growing evidences indicate that O-GlcNAcylation plays crucial roles in regulating immunity and inflammatory responses, especially under hyperglycemic conditions. This review will highlight the emerging functions of O-GlcNAcylation in mammalian immunity under physiological and various pathological conditions.
Subject(s)
Acylation/immunology , Immunity/immunology , N-Acetylglucosaminyltransferases/immunology , Animals , Humans , Inflammation/immunologyABSTRACT
Adjuvants are essential vaccine components used to enhance, accelerate, and/or prolong adaptive immunity against specific vaccine antigens. In this study, we compared the adjuvanticity of two adjuvant formulations containing de-O-acylated lipooligosaccharide (dLOS), a toll-like receptor 4 agonist, on the Japanese encephalitis (JE) vaccine in mice. Mice were immunized once or twice at a two-week interval with inactivated JE vaccine in the absence or presence of adjuvant. We found that both the alum- and the liposome-based formulation induced significantly faster and higher serum IgG antibody responses as compared with the non-adjuvanted vaccine after either one or two immunizations. The antibody titers of the mouse immune sera correlated with 50% plaque reduction neutralization test (PRNT50) antibody titers. In addition, the dLOS/liposome formulation was more effective in inducing a Th1-type immune response than the dLOS/alum formulation, as suggested by a strong antigen-specific interferon (IFN)-γ response. Based on these results, we suggest that both alum- and liposome-based adjuvant formulations containing dLOS may be used for the development of JE vaccines with improved immunogenicity.
Subject(s)
Adjuvants, Immunologic , Antigens, Bacterial/immunology , Japanese Encephalitis Vaccines/immunology , Lipopolysaccharides/immunology , Acylation/immunology , Adjuvants, Immunologic/blood , Animals , Antigens, Bacterial/blood , Drug Compounding , Female , Japanese Encephalitis Vaccines/blood , Lipopolysaccharides/blood , Mice , Mice, Inbred BALB C , Protein Binding/immunologyABSTRACT
Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with â¼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/immunology , Lipid A/immunology , Shigella/immunology , Toll-Like Receptor 2/immunology , Toll-Like Receptor 4/immunology , Acylation/immunology , Acyltransferases/genetics , Acyltransferases/metabolism , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cells, Cultured , Cytokines/immunology , Cytokines/metabolism , Electrophoresis, Polyacrylamide Gel , HEK293 Cells , Humans , Lipid A/analysis , Lipid A/metabolism , Microscopy, Electron, Transmission , Monocytes/immunology , Monocytes/metabolism , Mutation , Shigella/genetics , Shigella/metabolism , Shigella flexneri/genetics , Shigella flexneri/immunology , Shigella flexneri/metabolism , Shigella sonnei/genetics , Shigella sonnei/immunology , Shigella sonnei/metabolism , Signal Transduction/immunology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolismABSTRACT
Lipid A, the hydrophobic anchor of lipopolysaccharide (LPS), is an essential component in the outer membrane of Gram-negative bacteria. It can stimulate the innate immune system via Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD2), leading to the release of inflammatory cytokines. In this study, six Escherichia coli strains which can produce lipid A with different acylation patterns were constructed; the influence of lipid A acylation pattern on the membrane permeability and innate immune stimulation has been systematically investigated. The lipid A species were isolated and identified by matrix assisted laser ionization desorption-time of flight/tandem mass spectrometry. N-Phenyl naphthylamine uptake assay and antibiotic susceptibility test showed that membrane permeability of these strains were different. The lower the number of acyl chains in lipid A, the stronger the membrane permeability. LPS purified from these strains were used to stimulate human or mouse macrophage cells, and different levels of cytokines were induced. Compared with wild type hexa-acylated LPS, penta-acylated, tetra-acylated and tri-acylated LPS induced lower levels of cytokines. These results suggest that the lipid A acylation pattern influences both the bacterial membrane permeability and innate immune stimulation. The results would be useful for redesigning the bacterial membrane structure and for developing lipid A vaccine adjuvant.
Subject(s)
Acylation/immunology , Cell Membrane Permeability/immunology , Escherichia coli/immunology , Escherichia coli/metabolism , Immunity, Innate/immunology , Lipid A/immunology , Lipid A/metabolism , Animals , Cell Membrane/immunology , Cell Membrane/metabolism , Cytokines/immunology , Cytokines/metabolism , Humans , Lipopolysaccharides/immunology , Macrophages/immunology , Macrophages/metabolism , MiceABSTRACT
Much evidence indicates that bacterial LPS (endotoxin) is removed from the bloodstream mainly by the liver, yet the hepatic uptake mechanisms remain uncertain and controversial. In plasma, LPS can be either 'free' (as aggregates, bacterial membrane fragments or loosely bound to albumin, CD14, or other proteins) or 'bound' (complexed with lipoproteins). Whereas most free LPS is taken up by Kupffer cells (KCs), lipoprotein-bound LPS has seemed to be cleared principally by hepatocytes. Here, we compared the liver's ability to take up and deacylate free LPS aggregates and the LPS in preformed LPS-high density lipoprotein (HDL) complexes. In mice examined from 1 h to 7 d after a small amount of fluorescent (FITC-)LPS was injected into a lateral tail vein, we found FITC-LPS almost entirely within, or adjacent to, KCs. As expected, FITC-LPS complexed with HDL (FITC-LPS-HDL) disappeared more slowly from the circulation and a smaller fraction of the injected dose of FITC-LPS was found in the liver. Unexpectedly, the FITC-LPS injected as FITC-LPS-HDL complexes was also found within sinusoids, adjacent to, or within, KCs. In other experiments, we found that both free and HDL-bound radiolabeled LPS underwent enzymatic deacylation by acyloxyacyl hydrolase (AOAH), the LPS-inactivating enzyme that is principally produced within the liver by KCs. Our observations suggest that KCs and AOAH play important roles in clearing and catabolizing both free LPS and the LPS in circulating LPS-HDL complexes.