QcrC is a potential target for antibody therapy and vaccination to control Campylobacter jejuni infection by suppressing its energy metabolism.

Introduction: Campylobacter spp. are a public health concern, yet there is still no effective vaccine or medicine available. Methods: Here, we developed a Campylobacter jejuni-specific antibody and found that it targeted a menaquinol cytochrome c reductase complex QcrC. Results: The antibody was specifically reactive to multiple C. jejuni strains including clinical isolates from patients with acute enteritis and was found to inhibit the energy metabolism and growth of C. jejuni. Different culture conditions produced different expression levels of QcrC in C. jejuni, and these levels were closely related not only to the energy metabolism of C. jejuni but also its pathogenicity. Furthermore, immunization of mice with recombinant QcrC induced protective immunity against C. jejuni infection. Discussion: Taken together, our present findings highlight a possible antibody- or vaccination-based strategy to prevent or control Campylobacter infection by targeting the QcrC-mediated metabolic pathway.

The omega-3 postbiotic trans-10-cis-15-octadecadienoic acid attenuates contact hypersensitivity in mice through downregulation of vascular endothelial growth factor A.

Intestinal bacteria metabolize dietary substances to produce bioactive postbiotics, among which some are recognized for their role in promoting host health. We here explored the postbiotic potential of two omega-3 α-linolenic acid-derived metabolites: trans-10-cis-15-octadecadienoic acid (t10,c15-18:2) and cis-9-cis-15-octadecadienoic acid (c9,c15-18:2). Dietary intake of lipids rich in omega-3 α-linolenic acid elevated levels of t10,c15-18:2 and c9,c15-18:2 in the serum and feces of mice, an effect dependent on the presence of intestinal bacteria. Notably, t10,c15-18:2 mitigated skin inflammation in mice that became hypersensitive after exposure to 2,4-dinitrofluorobenzene, an experimental model for allergic contact dermatitis. In particular, t10,c15-18:2-but not c9,c15-18:2-attenuated ear swelling and edema, characteristic symptoms of contact hypersensitivity. The anti-inflammatory effects of t10,c15-18:2 were due to its ability to suppress the release of vascular endothelial growth factor A from keratinocytes, thereby mitigating the enhanced vascular permeability induced by hapten stimulation. Our study identified retinoid X receptor as a functional receptor that mediates the downregulation of skin inflammation upon treatment with t10,c15-18:2. Our results suggest that t10,c15-18:2 holds promise as an omega-3 fatty acid-derived postbiotic with potential therapeutic implications for alleviating the skin edema seen in allergic contact dermatitis-induced inflammation.

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 nonhomologous 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.

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.

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.

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.

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.

Structural identification of catalytic His158 of PtMAC2p from Pseudozyma tsukubaensis, an acyltransferase involved in mannosylerythritol lipids formation.

Mannosylerythritol lipids (MELs) are extracellular glycolipids produced by the basidiomycetous yeast strains. MELs consist of the disaccharide mannosylerythritol, which is acylated with fatty acids and acetylated at the mannose moiety. In the MEL biosynthesis pathway, an acyltransferase from Pseudozyma tsukubaensis, PtMAC2p, a known excellent MEL producer, has been identified to catalyze the acyl-transfer of fatty acid to the C3'-hydroxyl group of mono-acylated MEL; however, its structure remains unclear. Here, we performed X-ray crystallography of recombinant PtMAC2p produced in Escherichia coli and homogeneously purified it with catalytic activity in vitro. The crystal structure of PtMAC2p was determined by single-wavelength anomalous dispersion using iodide ions. The crystal structure shows that PtMAC2p possesses a large putative catalytic tunnel at the center of the molecule. The structural comparison demonstrated that PtMAC2p is homologous to BAHD acyltransferases, although its amino acid-sequence identity was low (<15%). Interestingly, the HXXXD motif, which is a conserved catalytic motif in the BAHD acyltransferase superfamily, is partially conserved as His158-Thr159-Leu160-Asn161-Gly162 in PtMAC2p, i.e., D in the HXXXD motif is replaced by G in PtMAC2p. Site-directed mutagenesis of His158 to Ala resulted in more than 1,000-fold decrease in the catalytic activity of PtMAC2p. These findings suggested that His158 in PtMAC2p is the catalytic residue. Moreover, in the putative catalytic tunnel, hydrophobic amino acid residues are concentrated near His158, suggesting that this region is a binding site for the fatty acid side chain of MEL (acyl acceptor) and/or acyl-coenzyme A (acyl donor). To our knowledge, this is the first study to provide structural insight into the catalytic activity of an enzyme involved in MEL biosynthesis.

Induction of unique macrophage subset by simultaneous stimulation with LPS and IL-4.

Macrophages manifest as various subtypes that play diverse and important roles in immunosurveillance and the maintenance of immunological homeostasis in various tissues. Many in vitro studies divide macrophages into two broad groups: M1 macrophages induced by lipopolysaccharide (LPS), and M2 macrophages induced by interleukin 4 (IL-4). However, considering the complex and diverse microenvironment in vivo, the concept of M1 and M2 is not enough to explain diversity of macrophages. In this study, we analyzed the functions of macrophages induced by simultaneous stimulation with LPS and IL-4 (termed LPS/IL-4-induced macrophages). LPS/IL-4-induced macrophages were a homogeneous population showing a mixture of the characteristics of M1 and M2 macrophages. In LPS/IL-4-induced macrophages, expression of cell-surface M1 markers (I-Ab) was higher than in M1 macrophages, but lower expression of iNOS, and expression of M1-associated genes (Tnfα and Il12p40) were decreased in comparison to expression in M1 macrophages. Conversely, expression of the cell-surface M2 marker CD206 was lower on LPS/IL-4-induced macrophages than on M2 macrophages and expression of M2-associated genes (Arg1, Chi3l3, and Fizz1) varied, with Arg1 being greater than, Fizz1 being lower than, and Chi3l3 being comparable to that in M2 macrophages. Glycolysis-dependent phagocytic activity of LPS/IL-4-induced macrophages was strongly enhanced as was that of M1 macrophages; however, the energy metabolism of LPS/IL-4-induced macrophages, such as activation state of glycolytic and oxidative phosphorylation, was quite different from that of M1 or M2 macrophages. These results indicate that the macrophages induced by LPS and IL-4 had unique properties.