Macrophage migration inhibitory factor-mediated mast cell extracellular traps induce inflammatory responses upon Fusobacterium nucleatum infection.

Mast cell extracellular traps (MCETs) released by mast cells contribute to host defense. In this study, we investigated the effects of MCETs released from mast cells after infection with a periodontal pathogen Fusobacterium nucleatum. We found that F. nucleatum induced MCET release from mast cells, and that MCETs expressed macrophage migration inhibitory factor (MIF). Notably, MIF bound to MCETs induced proinflammatory cytokine production by monocytic cells. These findings suggest that MIF expressed on MCETs, released from mast cells upon infection with F. nucleatum, promotes inflammatory responses that may be associated with the pathogenesis of periodontal disease.

Comparative analysis of glyceroglycolipids from Lactiplantibacillus pentosus and other Lactiplantibacillus species.

Cellular lipids of Lactiplantibacillus species were extracted and neutral glyceroglycolipids (GGLs) were purified, and analyzed by thin-layer chromatography (TLC). Four GGLs with known structures were detected in GGL preparation of L. plantarum, and the same GGL profiles of TLC were observed for all other strains of Lactiplantibacillus species examined, suggesting that the GGL profile could be one of the chemotaxonomic characters of the genus Lactiplantibacillus. On the other hand, the quantity of each GGL showed some variation among species, and L. pentosus was found to have higher proportion of disaccharide-type GGL, designated GGL-3 in this study, compared with other species including L. plantarum. The quantitative difference of GGL-3 found in this study could be regarded as the characteristics of L. pentosus. The carbohydrate structure of L. pentosus GGL-3 was precisely analyzed by 1H NMR and methylation analysis, and the structure was confirmed to be αGal-(1→2)-αGlc-diacylglycerol, with the carbohydrate structure identical to that of L. plantarum, although fatty acid composition of the two GGL-3 showed some difference.

Hericium erinaceus ethanol extract and ergosterol exert anti-inflammatory activities by neutralizing lipopolysaccharide-induced pro-inflammatory cytokine production in human monocytes.

Edible mushrooms are known to exert anti-inflammatory effects. In this study, the effects of ethanol extracts from edible mushrooms, such as Hericium erinaceus, and other edible mushrooms on inflammatory responses were investigated. Experiments were conducted using the inflammatory responses of human monocytes induced by lipopolysaccharide (LPS), a bacterial component, that provokes inflammation. Notably, we demonstrated that LPS mixed with ethanol and hot water extracts derived from edible mushrooms attenuated the production of inflammatory cytokines, such as interleukin (IL)-1ß, -6, and -8, induced by LPS in human monocytic cell cultures. Moreover, we found that the ethanol extract of H. erinaceus contained ergosterol, which attenuated IL-8 production in LPS-stimulated cells. Subsequent component analysis of the ethanol extract of H. erinaceus revealed that ergosterol binds to lipid A to attenuate LPS-induced inflammation. Together, our findings suggest that ergosterol in ethanol extracts from edible mushrooms can prevent the induction of inflammation by binding to LPS.

A highly immunogenic vaccine platform against encapsulated pathogens using chimeric probiotic Escherichia coli membrane vesicles.

Vaccines against infectious diseases should elicit potent and long-lasting immunity, ideally even in those with age-related decline in immune response. Here we report a rational polysaccharide vaccine platform using probiotic Escherichia coli-derived membrane vesicles (MVs). First, we constructed a probiotic E. coli clone harboring the genetic locus responsible for biogenesis of serotype 14 pneumococcal capsular polysaccharides (CPS14) as a model antigen. CPS14 was found to be polymerized and mainly localized on the outer membrane of the E. coli cells. The glycine-induced MVs displayed the exogenous CPS14 at high density on the outermost surface, on which the CPS14 moiety was covalently tethered to a lipid A-core oligosaccharide anchor. In in vivo immunization experiments, CPS14+MVs, but not a mixture of free CPS14 and empty MVs, strongly elicited IgG class-switch recombination with a Th1/Th2-balanced IgG subclass distribution without any adjuvant. In addition, CPS14+MVs were structurally stable with heat treatment and immunization with the heat-treated MVs-elicited CPS14-specific antibody responses in mouse serum to levels comparable to those of non-treated CPS14+MVs. Notably, the immunogenicity of CPS14+MVs was significantly stronger than those of two currently licensed vaccines against pneumococci. The CPS14+MV-elicited humoral immune responses persisted for 1 year in both blood and lung. Furthermore, the CPS14+MV vaccine was widely efficacious in mice of different ages. Even in aged mice, vaccination resulted in robust production of CPS14-specific IgG that bound to the pneumococcal cell surface. Taken together, the present probiotic E. coli MVs-based vaccine platform offers a promising, generalizable solution against encapsulated pathogens.

Chemical characterization of ceramides isolated from a gliding marine bacterium Aureispira marina.

Ceramides containing 2-hydroxy fatty acids were purified from a gliding marine bacterium Aureispira marina, and their chemical structure was investigated. The ceramide molecules contained 2-hydroxy-15-methyl-hexadecanoic acid and 2-hydroxy-15-methyl-hexadecenoic acid, and the double bond of the latter fatty acid was proved to be located between the positions C3 and C4. The major portion of these 2-hydroxy fatty acids was determined to have D-configuration (S-configuration) after diastereomeric derivatization. Three carbon skeletons were found in sphingosines from ceramides, ie (1) 1,3-dihydroxy-2-amino-4-octadecen, (2) 1,3-dihydroxy-2-amino-17-methyl-4-octadecen, and (3) 1,3-dihydroxy-2-amino-9-methyl-4-octadecen. Molecules with additional double bonds were found in sphingosines with structures 1 and 3. The presence of ceramides with these chemical characteristics would be a significant feature for the taxonomy of A. marina and related bacteria.

Detection of 2-hydroxy-fatty acids and 2-hydroxy-fatty acid-containing ceramides in a gliding marine bacterium Aureispira marina.

The cellular fatty acid composition of Aureispira marina IAM 15389T (JCM 23197T), a gliding bacterium isolated from the coastline of Thailand, was re-examined by using a standard MIDI method based on alkaline hydrolysis, and two other methods. The direct transesterification using 5% HCl/methanol or 4 M HCl hydrolysis followed by methyl esterification revealed that 2-hydroxy-15-methyl-hexadecanoic acid (2-OH-iso-C17:0) and 2-hydroxy-15-methyl-hexadecenoic acid (2-OH-iso-C17:1), which were not reported in a previous paper, were found to be major cellular fatty acids of this bacterium, and the amount of 2-OH-iso-C17:1 was even higher than that of arachidonic acid (C20:4), a characteristic polyunsaturated fatty acid present in this bacterium. These 2-hydroxy-fatty acids were contained in two cellular lipids that were relatively stable against alkaline hydrolysis. One of them was analyzed by mass spectrometry, 1H-nuclear magnetic resonance, and other chemical methods, and identified as a ceramide composed of 2-hydroxy-fatty acid and sphingosine of 19 carbons with three double bonds. A minor ceramide containing 18 carbon sphingosine with three double bonds was also detected.

Variation, Modification and Engineering of Lipid A in Endotoxin of Gram-Negative Bacteria.

Lipid A of Gram-negative bacteria is known to represent a central role for the immunological activity of endotoxin. Chemical structure and biosynthetic pathways as well as specific receptors on phagocytic cells had been clarified by the beginning of the 21st century. Although the lipid A of enterobacteria including Escherichia coli share a common structure, other Gram-negative bacteria belonging to various classes of the phylum Proteobacteria and other taxonomical groups show wide variety of lipid A structure with relatively decreased endotoxic activity compared to that of E. coli. The structural diversity is produced from the difference of chain length of 3-hydroxy fatty acids and non-hydroxy fatty acids linked to their hydroxyl groups. In some bacteria, glucosamine in the backbone is substituted by another amino sugar, or phosphate groups bound to the backbone are modified. The variation of structure is also introduced by the enzymes that can modify electrostatic charges or acylation profiles of lipid A during or after its synthesis. Furthermore, lipid A structure can be artificially modified or engineered by the disruption and introduction of biosynthetic genes especially those of acyltransferases. These technologies may produce novel vaccine adjuvants or antagonistic drugs derived from endotoxin in the future.

Structural modification of Escherichia coli lipid A by myristoyltransferase gene from Klebsiella pneumoniae.

Lipid A in lipopolysaccharide (LPS) of Escherichia coli mutant strains was modified by the introduction of myristoyltransferase gene cloned from Klebsiella pneumoniae. When the gene was introduced into the mutant having lipid A containing only 3-hydroxymyristic acids, it produced lipid A with two additional myristic acids (C14:0 ). When the same gene was introduced into the mutant with pentaacylated lipid A containing one lauric acid (C12:0 ), C12:0 was replaced by C14:0 . IL-6-inducing activity of LPS with modified lipid A structure suggested that C12:0 in lipid A could be replaced by C14:0 without changing the immunostimulating activity.

Synthetic chemistry with friendships that unveiled the long-lasting mystery of lipid A.

Endotoxin research in recent years at the molecular level has required chemically synthesized lipid A without contamination by other bioactive components. Total synthesis of Escherichia coli-type lipid A was achieved in the 1980s by the challenging spirits of the scientists at Osaka University, Japan. They clarified the role of lipid A in the immunological activities of endotoxin in collaboration with Japanese and German researchers, based on the friendships that existed between them. This article introduces the great contributions made by three generations of professors, Tetsuo Shiba, Shoichi Kusumoto, and Koichi Fukase, at the Laboratory of Natural Product Chemistry at Osaka University, to the study over four decades of endotoxin.

Modification of lipid A structure and activity by the introduction of palmitoyltransferase gene to the acyltransferase-knockout mutant of Escherichia coli.

Lauroyltransferase gene (lpxL), Myristoyltransferase gene (lpxM) and palmitoyltransferase gene (crcA) of Escherichia coli BL21 were independently disrupted by the insertional mutations. The knockout mutant of two transferase genes (lpxL and crcA) produced lipid A with no lauric or palmitic acids and only a little amount of myristic acid. The mutant was susceptible to polymyxin B, but showed comparable growth with the wild-type strain at 30°C. The palmitoyltransferase gene from E. coli (crcA) or Salmonella (pagP) was amplified by PCR, cloned in pUC119, and transferred into the double-knockout mutant by transformation. The transformant contained palmitic acid in the lipid A, and recovered resistance to polymyxin B. Mass spectrometric analysis revealed that palmitic acid was linked to the hydroxyl group of 3-hydroxymyristic acid at C-2 position of proximal (reducing-end) glucosamine. LPS from the double-knockout mutant showed reduced IL-6-inducing activity to macrophage-like line cells compared to that of the wild-type strain, and the activity was only slightly restored by the introduction of palmitic acid to the lipid A. These results suggested that the introduction of one palmitic acid was enough to recover the integrity of the outer membrane, but not enough for the stimulation of macrophages.

Determination of O:4 antigen-antibody affinity level in O:5 antigen positive and negative variants of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) has two serological variants: one that expresses the O:5 antigen (1,4,5,12:i:1,2) and one that lacks O:5 antigen (1,4,12:i:1,2). For serotyping, S. Typhimurium is agglutinated by diagnostic O:4 antigen serum. This study was carried out to compare the antigen-antibody affinity of O:4 antigen in S. Typhimurium χ3306 O:5-positive and S. Typhimurium χ3306 O:5-negative strains. The affinity of O:4 antigen with O:4 antigen serum was found to be stronger in the O:5-negative strains compared to O:5-positive strains. Next, we investigated the antigen-antibody affinity of O:4 antigen with O:4 antigen serum in field strains of S. Typhimurium, which showed the same tendency in affinity as seen with S. Typhimurium χ3306 O:5-positive and negative strains. This study suggests that the presence or absence of O:5 antigen causes differences in O:4 agglutination reactions with different field strains of S. Typhimurium.

Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme, Sphingobium fuliginis ATCC 27551.

The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.

KLRG+ invariant natural killer T cells are long-lived effectors.

Immunological memory has been regarded as a unique feature of the adaptive immune response mediated in an antigen-specific manner by T and B lymphocytes. However, natural killer (NK) cells and γδT cells, which traditionally are classified as innate immune cells, have been shown in recent studies to have hallmark features of memory cells. Invariant NKT cell (iNKT cell)-mediated antitumor effects indicate that iNKT cells are activated in vivo by vaccination with iNKT cell ligand-loaded CD1d(+) cells, but not by vaccination with unbound NKT cell ligand. In such models, it previously was thought that the numbers of IFN-γ-producing cells in the spleen returned to the basal level around 1 wk after the vaccination. In the current study, we demonstrate the surprising presence of effector memory-like iNKT cells in the lung. We found long-term antitumor activity in the lungs of mice was enhanced after vaccination with iNKT cell ligand-loaded dendritic cells. Further analyses showed that the KLRG1(+) (Killer cell lectin-like receptor subfamily G, member 1-positive) iNKT cells coexpressing CD49d and granzyme A persisted for several months and displayed a potent secondary response to cognate antigen. Finally, analyses of CDR3ß by RNA deep sequencing demonstrated that some particular KLRG1(+) iNKT-cell clones accumulated, suggesting the selection of certain T-cell receptor repertoires by an antigen. The current findings identifying effector memory-like KLRG1(+) iNKT cells in the lung could result in a paradigm shift regarding the basis of newly developed extrathymic iNKT cells and could contribute to the future development of antitumor immunotherapy by uniquely energizing iNKT cells.

Interactions of glycosphingolipids and lipopolysaccharides with silica and polyamide surfaces: adsorption and viscoelastic properties.

Bacterial outer membrane components play a critical role in bacteria-surface interactions (adhesion and repulsion). Sphingomonas species (spp.) differ from other Gram-negative bacteria in that they lack lipopolysaccharides (LPSs) in their outer membrane. Instead, Sphingomonas spp. outer membrane consists of glycosphingolipids (GSLs). To delineate the properties of the outer membrane of Sphingomonas spp. and to explain the adhesion of these cells to surfaces, we employed a single-component-based approach of comparing GSL vesicles to LPS vesicles. This is the first study to report the formation of vesicles containing 100% GSL. Significant physicochemical differences between GSL and LPS vesicles are reported. Composition-dependent vesicle adherence to different surfaces using quartz crystal microbalance with dissipation monitoring (QCM-D) technology was observed, where higher GSL content resulted in higher mass accumulation on the sensor. Additionally, the presence of 10% GSL and above was found to promote the relative rigidity of the vesicle obtaining viscoelastic ratio of 30-70% higher than that of pure LPS vesicles.

Isolation and structural characterization of a (Kdo-isosteric) D-glycero-α-D-talo-oct-2-ulopyranosidonic acid (Ko) interlinking lipid A and core oligosaccharide in the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305.

In Acinetobacter calcoaceticus NCTC 10305 and A. haemolyticus NCTC 10305 lipopolysaccharide (LPS) a Kdo (3-deoxy-D-manno-oct-2-ulosonic acid)-related octulosonic acid (Ko) interlinks the lipid A with the core-oligosaccharide. This Ko replaces the first Kdo (Kdo(I)) attached to the lipid A backbone in the LPS. The only structural difference between Kdo and Ko is the 3-hydroxylation. After the discovery of the final step in Ko-biosynthesis it is now generally accepted that Ko is structurally related to Kdo, although a final proof so far is lacking. In the present paper we describe the stereochemical determination of the natural Ko isolated from the LPS of A. calcoaceticus NCTC 10305 by chemical, mass spectrometry (MS), and (1)H and (13)C NMR spectroscopy. Our results show that in A. calcoaceticusd-glycero-α-D-talo-oct-2-ulopyranosonic acid (DgαDt-Kop) represents the Kdo-related sugar interlinking the core-oligosaccharide and the lipid A backbone.

CD1d protein structure determines species-selective antigenicity of isoglobotrihexosylceramide (iGb3) to invariant NKT cells.

Isoglobotrihexosylceramide (iGb3) has been identified as a potent CD1d-presented self-antigen for mouse invariant natural killer T (iNKT) cells. The role of iGb3 in humans remains unresolved, however, as there have been conflicting reports about iGb3-dependent human iNKT-cell activation, and humans lack iGb3 synthase, a key enzyme for iGb3 synthesis. Given the importance of human immune responses, we conducted a human-mouse cross-species analysis of iNKT-cell activation by iGb3-CD1d. Here we show that human and mouse iNKT cells were both able to recognise iGb3 presented by mouse CD1d (mCD1d), but not human CD1d (hCD1d), as iGb3-hCD1d was unable to support cognate interactions with the iNKT-cell TCRs tested in this study. The structural basis for this discrepancy was identified as a single amino acid variation between hCD1d and mCD1d, a glycine-to-tryptophan modification within the α2-helix that prevents flattening of the iGb3 headgroup upon TCR ligation. Mutation of the human residue, Trp153, to the mouse ortholog, Gly155, therefore allowed iGb3-hCD1d to stimulate human iNKT cells. In conclusion, our data indicate that iGb3 is unlikely to be a major antigen in human iNKT-cell biology.

Sphingobacterium thermophilum sp. nov., of the phylum Bacteroidetes, isolated from compost.

A Gram-negative bacterium, designated CKTN2(T), was isolated from compost. Cells of strain CKTN2(T) were strictly aerobic rods. The isolate grew at 20-50 °C (optimum 40-45 °C), but not below 15 °C or above 52 °C, and at pH 5.9-8.8 (optimum pH 7.0), but not below pH 5.4 or above pH 9.3. The DNA G+C content was 40.3 mol%. The predominant menaquinone was MK-7. The major fatty acids were iso-C15 : 0 (45.2 %), iso-C17 : 0 3-OH (11.1 %) and C18 : 0 (14.5 %). Analysis of the 16S rRNA gene sequence of strain CKTN2(T) revealed that it is a member of the genus Sphingobacterium and is most closely related to Sphingobacterium alimentarium DSM 22362(T) (93.2 % 16S rRNA gene sequence similarity). Strain CKTN2(T) could be distinguished from its closest phylogenetic relatives by different phenotypic characteristics. According to the phenotypic and genotypic characteristics, strain CKTN2(T) represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium thermophilum sp. nov. is proposed. The type strain is CKTN2(T) ( = JCM 17858(T)  = KCTC 23708(T)).

Regulation of memory CD4 T-cell pool size and function by natural killer T cells in vivo.

To develop more effective vaccines and strategies to regulate chronic inflammatory diseases, it is important to understand the mechanisms of immunological memory. Factors regulating memory CD4(+) T helper (Th)-cell pool size and function remain unclear, however. We show that activation of type I invariant natural killer T (iNKT) cells with glycolipid ligands and activation of type II natural killer T (NKT) cells with the endogenous ligand sulfatide induced dramatic proliferation and expansion of memory, but not naïve, CD4 T cells. NKT cell-induced proliferation of memory Th1 and Th2 cells was dependent largely on the production of IL-2, with Th2-cell proliferation also affected by loss of IL-4. Type II NKT cells were also required for efficient maintenance of memory CD4 T cells in vivo. Activation of iNKT cells resulted in up-regulation of IFN-γ expression by memory Th2 cells. These IFN-γ-producing memory Th2 cells showed a decreased capability to induce Th2 cytokines and eosinophilic airway inflammation. Thus, activated NKT cells directly regulate memory CD4 T-cell pool size and function via the production of cytokines in vivo.

Evasion of human innate immunity without antagonizing TLR4 by mutant Salmonella enterica serovar Typhimurium having penta-acylated lipid A.

Modification of a lipid A moiety in Gram-negative bacterial LPS to a less acylated form is thought to facilitate bacterial evasion of host innate immunity, thereby enhancing pathogenicity. The contribution of less-acylated lipid A to interactions of whole bacterial cells with host cells (especially in humans) remains unclear. Mutant strains of Salmonella enterica serovar Typhimurium with fewer acylated groups were generated. The major lipid A form in wild-type (WT) and the mutant KCS237 strain is hexa-acylated; in mutant strains KCS311 and KCS324 it is penta-acylated; and in KCS369 it is tetra-acylated. WT and KCS237 formalin-killed and live bacteria, as well as their LPS, strongly stimulated production of pro-inflammatory cytokines in human U937 cells; this stimulation was suppressed by TLR4 suppressors. LPS of other mutants produced no agonistic activity, but strong antagonistic activity, while their formalin-killed and live bacteria preparations had weak agonistic and no antagonistic activity. Moreover, these less-acylated mutants had increased resistance to phagocytosis by U937 cells. Our results indicate that a decrease of one acyl group (from six to five) is enough to allow Salmonella to evade human innate immunity and that the antagonistic activity of less-acylated lipid A is not utilized for this evasion.