Alcaligenes lipid A functions as a superior mucosal adjuvant to monophosphoryl lipid A via the recruitment and activation of CD11b+ dendritic cells in nasal tissue.

We previously demonstrated that Alcaligenes-derived lipid A (ALA), which is produced from an intestinal lymphoid tissue-resident commensal bacterium, is an effective adjuvant for inducing antigen-specific immune responses. To understand the immunologic characteristics of ALA as a vaccine adjuvant, we here compared the adjuvant activity of ALA with that of a licensed adjuvant (monophosphoryl lipid A, MPLA) in mice. Although the adjuvant activity of ALA was only slightly greater than that of MPLA for subcutaneous immunization, ALA induced significantly greater IgA antibody production than did MPLA during nasal immunization. Regarding the underlying mechanism, ALA increased and activated CD11b+ CD103- CD11c+ dendritic cells in the nasal tissue by stimulating chemokine responses. These findings revealed the superiority of ALA as a mucosal adjuvant due to the unique immunologic functions of ALA in nasal tissue.

Linkage-Editing Pseudo-Glycans: A Reductive α-Fluorovinyl-C-Glycosylation Strategy to Create Glycan Analogs with Altered Biological Activities.

The acetal (O-glycoside) bonds of glycans and glycoconjugates are chemically and biologically vulnerable, and therefore C-glycosides are of interest as more stable analogs. We hypothesized that, if the O-glycoside linkage plays a vital role in glycan function, the biological activities of C-glycoside analogs would vary depending on their substituents. Based on this idea, we adopted a "linkage-editing strategy" for the creation of glycan analogs (pseudo-glycans). We designed three types of pseudo-glycans with CH2 and CHF linkages, which resemble the O-glycoside linkage in terms of bond lengths, angles, and bulkiness, and synthesized them efficiently by means of fluorovinyl C-glycosylation and selective hydrogenation reactions. Application of this strategy to isomaltose (IM), an inducer of amylase expression, and α-GalCer, which activates iNKT cells, resulted in the discovery of CH2-IM, which shows increased amylase production ability, and CHF-α-GalCer, which shows activity opposite that of native α-GalCer, serving as an antagonist of iNKT cells.

Characterization of a KU70-disrupted strain of the mannosylerythritol lipid-producing yeast Pseudozyma tsukubaensis constructed by a marker recycling system.

The basidiomycetous yeast Pseudozyma tsukubaensis is known as an industrial mannosylerythritol lipid producer. In this study, the PtURA5 marker gene was deleted by homologous recombination. Using the PtURA5-deleted mutant as a host strain, we obtained a derivative disrupted for the PtKU70 gene, a putative ortholog of the KU70 gene encoding a protein involved in the non-homologous end-joining pathway of DNA repair. Subsequently, the introduced PtURA5 gene was re-deleted by marker recycling. These results demonstrated that the PtURA5 gene can be used as a recyclable marker gene. Although the frequency of homologous recombination has been shown to be increased by KU70 disruption in other fungi, the PtKU70-disrupted strain of P. tsukubaensis did not demonstrate an elevated frequency of homologous recombination. Furthermore, the PtKU70-disrupted strain did not show increased susceptibility to bleomycin. These results suggested that the function of this KU70 ortholog in P. tsukubaensis is distinct from that in other fungi.

Self-Assembling Properties and Recovery Effects on Damaged Skin Cells of Chemically Synthesized Mannosylerythritol Lipids.

Mannosylerythritol lipids (MELs), which are one of the representative sugar-based biosurfactants (BSs) produced by microorganisms, have attracted much attention in various fields in the sustainable development goals (SDGs) era. However, they are inseparable mixtures with respect to the chain length of the fatty acids. In this study, self-assembling properties and structure-activity relationship (SAR) studies of recovery effects on damaged skin cells using chemically synthesized MELs were investigated. It was revealed, for the first time, that synthetic and homogeneous MELs exhibited significant self-assembling properties to form droplets or giant vesicles. In addition, a small difference in the length of the fatty acid chains of the MELs significantly affected their recovery effects on the damaged skin cells. MELs with medium or longer length alkyl chains exhibited much higher recovery effects than that of C18-ceramide NP.

Synthesis of Mannosylerythritol Lipid Analogues and their Self-Assembling Properties, Recovery Effects on Damaged Skin Cells, and Antibacterial Activity.

Synthesis of three types of purpose-designed mannosylerythritol lipid (MEL)-D analogues with decanoyl groups, ß-GlcEL-D, α-GlcEL-D, and α-MEL-D, was accomplished utilizing our boron-mediated aglycon delivery (BMAD) methods. Their self-assembling properties, recovery effects on damaged skin cells, and antibacterial activity were evaluated. It was revealed, for the first time, that α-GlcEL-D and α-MEL-D only generated giant vesicles, indicating that slight differences in the steric configuration of an erythritol moiety and fatty acyl chains affect the ability to form vesicles. Analogue α-MEL-D exhibited significant recovery effects on damaged skin cells. Furthermore, α-MEL-D exhibited antibacterial activity as high as that for MEL-D, indicating that α-MEL-D is a promising artificial sugar-based material candidate for enhancing the barrier function of the stratum corneum, superior to a known cosmetic ingredient, and possesses antibacterial activity.

Selective expression of claudin-5 in thymic endothelial cells regulates the blood-thymus barrier and T-cell export.

T-cell development depends on the thymic microenvironment, in which endothelial cells (ECs) play a vital role. Interestingly, vascular permeability of the thymic cortex is lower than in other organs, suggesting the existence of a blood-thymus barrier (BTB). On the other hand, blood-borne molecules and dendritic cells bearing self-antigens are accessible to the medulla, facilitating central tolerance induction, and continuous T-precursor immigration and mature thymocyte egress occur through the vessels at the cortico-medullary junction (CMJ). We found that claudin-5 (Cld5), a membrane protein of tight junctions, was expressed in essentially all ECs of the cortical vasculatures, whereas approximately half of the ECs of the medulla and CMJ lacked Cld5 expression. An intravenously (i.v.) injected biotin tracer hardly penetrated cortical Cld5+ vessels, but it leaked into the medullary parenchyma through Cld5- vessels. Cld5 expression in an EC cell line caused a remarkable increase in trans-endothelial resistance in vitro, and the biotin tracer leaked from the cortical vasculatures in Cldn5-/- mice. Furthermore, i.v.-injected sphingosine-1 phosphate distributed selectively into the medulla through the Cld5- vessels, probably ensuring the egress of CD3high mature thymocytes from Cld5- vessels at the CMJ. These results suggest that distinct Cld5 expression profiles in the cortex and medulla may control the BTB and the T-cell gateway to blood circulation, respectively.

ω3 fatty acid metabolite, 12-hydroxyeicosapentaenoic acid, alleviates contact hypersensitivity by downregulation of CXCL1 and CXCL2 gene expression in keratinocytes via retinoid X receptor α.

ω3 fatty acids show potent bioactivities via conversion into lipid mediators; therefore, metabolism of dietary lipids is a critical determinant in the properties of ω3 fatty acids in the control of allergic inflammatory diseases. However, metabolic progression of ω3 fatty acids in the skin and their roles in the regulation of skin inflammation remains to be clarified. In this study, we found that 12-hydroxyeicosapentaenoic acid (12-HEPE), which is a 12-lipoxygenase metabolite of eicosapentaenoic acid, was the prominent metabolite accumulated in the skin of mice fed ω3 fatty acid-rich linseed oil. Consistently, the gene expression levels of Alox12 and Alox12b, which encode proteins involved in the generation of 12-HEPE, were much higher in the skin than in the other tissues (eg, gut). We also found that the topical application of 12-HEPE inhibited the inflammation associated with contact hypersensitivity by inhibiting neutrophil infiltration into the skin. In human keratinocytes in vitro, 12-HEPE inhibited the expression of two genes encoding neutrophil chemoattractants, CXCL1 and CXCL2, via retinoid X receptor α. Together, the present results demonstrate that the metabolic progression of dietary ω3 fatty acids differs in different organs, and identify 12-HEPE as the dominant ω3 fatty acid metabolite in the skin.

Evaluating haloarchaeal culture media for ultrahigh-molecular-weight polyhydroxyalkanoate biosynthesis by Haloferax mediterranei.

A series of culture media for haloarchaea were evaluated to optimize the production of ultrahigh-molecular-weight (UHMW) poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei. Cells of H. mediterranei grew (> 1 g/L of dry cell weight) and accumulated PHBV upon flask cultivation in 10 medium types with neutral pH and NaCl concentration > 100 g/L. Molecular weight and compositional analysis revealed that the number-average molecular weight (Mn) of PHBV produced with six selected types of media ranged from 0.8 to 3.5 × 106 g/mol and the 3-hydroxyvalerate (3HV) composition ranged from 8 to 36 mol%. Cultivation in two NBRC media, 1214 and 1380, resulted in the production of PHBV with an Mn of more than 3.0 × 106 g/mol and a weight-average molecular weight of more than 5.0 × 106 g/mol, indicating the production of UHMW-PHBV. These culture media contained small amount of complex nutrients like yeast extract and casamino acids, suggesting that H. mediterranei likely produced UHMW-PHBV on poor nutrient condition. Haloferax mediterranei grown in NBRC medium 1380 produced PHBV with the highest 3HV composition. A solvent-cast film of UHMW-PHBV with 26.4 mol% 3HV produced from 1-L flask cultivation with NBRC medium 1380 was found to be flexible and semi-transparent. Thermal analysis of the UHMW-PHBV cast film revealed melting and glass-transition temperatures of 90.5 °C and - 2.7 °C, respectively. KEY POINTS: • Haloarchaeal culture media were evaluated to produce UHMW-PHBV by H. mediterranei. • UHMW-PHBV with varied molecular weight was produced dependent on culture media. • Semi-transparent film could be made from UHMW-PHBV with 26.4 mol% 3HV.

Mechanisms underlying enhanced IgA production in Peyer's patch cells by membrane vesicles derived from Lactobacillus sakei.

We analyzed the mechanisms underlying enhanced IgA production in the cells of Peyer's patch cells via membrane vesicles derived from Lactobacillus sakei subsp. sakei NBRC 15893. Depletion of CD11c+ cells from Peyer's patch cells suppressed the enhanced IgA production mediated by membrane vesicles. Meanwhile, the stimulation of bone-marrow-derived dendritic cells with membrane vesicles increased gene expression of inducible nitric oxide synthase, retinaldehyde dehydrogenase 2, and several inflammatory cytokines. The production of nitric oxide and interleukin (IL)-6 by membrane vesicle stimulation was induced via Toll-like receptor 2 on bone marrow-derived dendritic cells. Inhibition of inducible nitric oxide synthase and retinaldehyde dehydrogenase 2, as well as the neutralization of IL-6 in Peyer's patch cells, suppressed the enhanced IgA production by membrane vesicle stimulation. Hence, nitric oxide, retinoic acid, and IL-6 induced by membrane vesicles play crucial roles in the enhanced IgA production elicited by membrane vesicles in Peyer's patch cells.

A putative transporter gene PtMMF1-deleted strain produces mono-acylated mannosylerythritol lipids in Pseudozyma tsukubaensis.

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by various yeasts. Mmf1, a putative transporter of MELs, is conserved in the MEL biosynthesis gene clusters of diverse MEL producers, including the genera Ustilago, Pseudozyma, Moesziomyces, and Sporisorium. To clarify the function of Mmf1, we generated the gene-deleted strain of P. tsukubaensis ΔPtMMF1 and evaluated its MEL production. Using thin-layer chromatography analyses, we detected most MELs produced by ΔPtMMF1 in the culture supernatant. The spot size of diacylated MEL-B (the only product of the parental strain) was significantly smaller for strain ΔPtMMF1 than for the parental strain, and a mono-acylated MEL-D spot was detected. In addition, an unknown glycolipid was detected in the sample extracted from strain ΔPtMMF1. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses revealed that the unknown glycolipid was a novel MEL homologue, mono-acylated MEL-B. KEY POINTS: • P. tsukubaensis is able to secrete MELs without PtMMF1p. • Strain ΔPtMMF1 mainly produced mono-acylated MELs.

Dietary coconut oil ameliorates skin contact hypersensitivity through mead acid production in mice.

Coconut oil is used as a dietary oil worldwide, and its healthy effects are recognized by the fact that coconut oil is easy to digest, helps in weight management, increases healthy cholesterol, and provides instant energy. Although topical application of coconut oil is known to reduce skin infection and inflammation, whether dietary coconut oil has any role in decreasing skin inflammation is unknown. In this study, we showed the impact of dietary coconut oil in allergic skin inflammation by using a mouse model of contact hypersensitivity (CHS). Mice maintained on coconut oil showed amelioration of skin inflammation and increased levels of cis-5, 8, 11-eicosatrienoic acid (mead acid) in serum. Intraperitoneal injection of mead acid inhibited CHS and reduced the number of neutrophils infiltrating to the skin. Detailed mechanistic studies unveiled that mead acid inhibited the directional migration of neutrophils by inhibiting the filamentous actin polymerization and leukotriene B4 production required for secondary recruitment of neutrophils. Our findings provide valuable insights into the preventive roles of coconut oil and mead acid against skin inflammation, thereby offering attractive therapeutic possibilities.

Tailor-made mannosylerythritol lipids: current state and perspectives.

Mannosylerythritol lipids (MELs) are a type of glycolipid biosurfactant produced by basidiomycetous yeasts, most notably those belonging to the genera Pseudozyma and Ustilago. Mannosylerythritol lipids are environmentally friendly and possess many unique functions, such as gene delivery, bio-activation, and human skin repair, and thus have potential applications in cosmetic, pharmaceutical, agriculture, food, and environmental industries. However, MELs will require overcoming same issues related to the commercialization, e.g., expansion of the structure and function variety and cost reduction. In the past decade, various studies have attempted to tailor production of targeted MELs in order to expand the utility of these biosurfactants. Moreover, the rapid development of genomic sequencing techniques will enhance our ability to modify MEL producers. In this review, we focus on current research into the tailored production of MELs, including conventional and advanced approaches.

Biosynthesis of mono-acylated mannosylerythritol lipid in an acyltransferase gene-disrupted mutant of Pseudozyma tsukubaensis.

The basidiomycetous yeast genus Pseudozyma produce large amounts of mannosylerythritol lipids (MELs), which are biosurfactants. A few Pseudozyma strains produce mono-acylated MEL as a minor compound using excess glucose as the sole carbon source. Mono-acylated MEL shows higher hydrophilicity than di-acylated MEL and has great potential for aqueous applications. Recently, the gene cluster involved in the MEL biosynthesis pathway was identified in yeast. Here, we generated an acyltransferase (PtMAC2) deletion strain of P. tsukubaensis 1E5 with uracil auxotrophy as a selectable marker. A PtURA5-mutant with a frameshift mutation in PtURA5 was generated as a uracil auxotroph of strain 1E5 by ultraviolet irradiation on plate medium containing 5-fluoro-orotic acid (5-FOA). In the mutant, PtMAC2 was replaced with a PtURA5 cassette containing the 5' untranslated region (UTR) (2000 bp) and 3' UTR (2000 bp) of PtMAC2 by homologous recombination, yielding strain ΔPtMAC2. Based on TLC and NMR analysis, we found that ΔPtMAC2 accumulates MEL acylated at the C-2' position of the mannose moiety. These results indicate that PtMAC2p catalyzes acylation at the C-3' position of the mannose of MEL.

Enhanced production of a diastereomer type of mannosylerythritol lipid-B by the basidiomycetous yeast Pseudozyma tsukubaensis expressing lipase genes from Pseudozyma antarctica.

Basidiomycetous yeasts in the genus Pseudozyma are known to produce extracellular glycolipids called mannosylerythritol lipids (MELs). Pseudozyma tsukubaensis produces a large amount of MEL-B using olive oil as the sole carbon source (> 70 g/L production). The MEL-B produced by P. tsukubaensis is a diastereomer type of MEL-B, which consists of 4-O-ß-D-mannopyranosyl-(2R,3S)-erythritol as a sugar moiety, in contrast to the conventional type of MELs produced by P. antarctica, which contain 4-O-ß-D mannopyranosyl-(2S,3R)-erythritol. In this study, we attempted to increase the production of the diastereomer type of MEL-B in P. tsukubaensis 1E5 by introducing the genes encoding two lipases, PaLIPAp (PaLIPA) and PaLIPBp (PaLIPB) from P. antarctica T-34. Strain 1E5 expressing PaLIPA exhibited higher lipase activity than the strain possessing an empty vector, which was used as a negative control. Strains of 1E5 expressing PaLIPA or PaLIPB showed 1.9- and 1.6-fold higher MEL-B production than the negative control strain, respectively, and oil consumption was also accelerated by the introduction of these lipase genes. MEL-B production was estimated using time course analysis in the recombinant strains. Strain 1E5 expressing PaLIPA produced 37.0 ± 1.2 g/L of MEL-B within 4 days of cultivation, whereas the strain expressing an empty vector produced 22.1 ± 7.5 g/L in this time. Overexpression of PaLIPA increased MEL-B production by P. tsukubaensis strain 1E5 from olive oil as carbon source by more than 1.7-fold.

Production of dicarboxylic acids from novel unsaturated fatty acids by laccase-catalyzed oxidative cleavage.

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon-carbon double bond were cleaved at the carbon-carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.

Genome-based analysis and gene dosage studies provide new insight into 3-hydroxy-4-methylvalerate biosynthesis in Ralstonia eutropha.

Recombinant Ralstonia eutropha strain PHB(-)4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB(-)4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB(-)4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone.

RNA-Based Anti-Inflammatory Effects of Membrane Vesicles Derived from Lactiplantibacillus plantarum.

Bacteria generally release extracellular membrane vesicles (MVs), which are nanoparticles that play important roles in bacterial-bacterial and bacterial-host communication. As probiotics, lactic acid bacteria provide diverse health benefits to their hosts. In this study, we found that the Gram-positive lactic acid bacteria Lactiplantibacillus plantarum subsp. plantarum NBRC 15891 produce high amounts of MVs (LpMVs), and that LpMVs inhibit interleukin (IL)-8 production induced by lipopolysaccharide in intestinal epithelial HT29 cells. Heat- or UV-killed bacterial cells did not exhibit anti-inflammatory effects, and there was no uptake of these bacterial cells; contrarily, LpMVs were taken up into the cytoplasm of HT29 cells. Small RNAs extracted from LpMVs also suppressed IL-8 production in HT29 cells, suggesting that RNAs in the cytoplasm of bacterial cells are encapsulated in the MVs and released from the cells, which may be delivered to HT29 cells to exert their anti-inflammatory effects. In addition, administration of LpMVs to mice with dextran sodium sulfate-induced colitis alleviated colitis-induced weight loss and colon length shortening, indicating that LpMV intake is likely to be effective in preventing or ameliorating colitis.

Biosynthetic ability of diverse basidiomycetous yeast strains to produce the natural antioxidant ergothioneine.

Sixteen strains of basidiomycetous yeasts were evaluated for their capability to produce ergothioneine (EGT), an amino acid derivative with strong antioxidant activity. The cells were cultured in either two synthetic media or yeast mold (YM) medium for 72 h, after which cytosolic constituents were extracted from the cells with hot water. After analyzing the extracts via liquid chromatography-mass spectrometry (LC-MS), we found that all strains produced varying amounts of EGT. The EGT-producing strains, including Ustilago siamensis, Anthracocystis floculossa, Tridiomyces crassus, Ustilago shanxiensis, and Moesziomyces antarcticus, were subjected to flask cultivation in YM medium. U. siamensis CBS9960 produced the highest amount of EGT at 49.5 ± 7.0 mg/L after 120 h, followed by T. crassus at 30.9 ± 1.8 mg/L. U. siamensis was also cultured in a jar fermenter and produced slightly higher amounts of EGT than under flask cultivation. The effects of culture conditions, particularly the addition of precursor amino acids, on EGT production by the selected strains were also evaluated. U. siamensis showed a 1.5-fold increase in EGT production with the addition of histidine, while U. shanxiensis experienced a 1.8-fold increase in EGT production with the addition of methionine. These results suggest that basidiomycetous yeasts could serve an abundant source for natural EGT producers.

High productivity of immunostimulatory membrane vesicles of Limosilactobacillus antri using glycine.

Nanosized membrane vesicles (MVs) released by bacteria play important roles in both bacteria-bacteria and bacteria-host interactions. Some gram-positive lactic acid bacteria produce MVs exhibiting immunoregulatory activity in the host. We found that both bacterial cells and MVs of Limosilactobacillus antri JCM 15950, isolated from the human stomach mucosa, enhance immunoglobulin A production by murine Peyer's patch cells. However, the thick cell walls of gram-positive bacteria resulted in low MV production, limiting experiments and applications using MVs. In this study, we evaluated the effects of glycine, which inhibits cell wall synthesis, on the immunostimulatory MV productivity of L. antri. Glycine inhibited bacterial growth while increasing MV production, with 20 g/L glycine increasing MV production approximately 12-fold. Glycine was most effective at increasing MV production when added in the early exponential phase, which indicated that cell division in the presence of glycine increased MV production. Finally, glycine increased MV productivity approximately 16-fold. Furthermore, glycine-induced MVs promoted interleukin-6 production by macrophage-like J774.1 cells, and the immunostimulatory activity was comparable to that of spontaneously produced MVs. Our results indicate that glycine is an effective agent for improving the production of MVs with immunostimulatory activity in gram-positive lactic acid bacteria, which can be applied as mucosal adjuvants and functional foods.