Bacterial glycolipids in Pediococcus pentosaceus for fermented soybean paste (miso) and Tetragenococcus halophilus for soy sauce

Bacterial glycolipids in Pediococcus pentosaceus for fermented soybean paste (miso) and Tetragenococcus halophilus for soy sauce | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bacterial glycolipids in Pediococcus pentosaceus for fermented soybean paste (miso) and Tetragenococcus halophilus for soy sauce Masao Iwamori, Kyoko Tanaka This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6265412/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jul, 2025 Read the published version in Glycoconjugate Journal → Version 1 posted 9 You are reading this latest preprint version Abstract Gram-positive bacterial biomembranes are composed of phosphatidyl glycerol (PG), cardiolipin (CL), and dihexaosyl diglycerides (DH-DG) as the major lipid constituents. The carbohydrate structures of DH-DG are specific to the particular bacterial species and we previously revealed them to have immunologically active properties. To characterize the functional significance of glycolipids in Gram-positive bacteria for fermented foods, the structures of DH-DG in Pediococcus pentosaceus (PP) for producing fermented soybean paste (miso) and Tetragenococcus halophilus (TH) for soy sauce were determined. They were shown to be Glcα1-2Glcα1-3′DG (kojibiosyl DG) with 18:1 and 18:1 for PP, and those with 16:0 and 18:1 for TH, both of which were identical to DH-DG from Streptococcus salivarius , a symbiotic bacterium in the human oral cavity. Additionally, both bacteria contained an acidic glycolipid, in which glycerol phosphate was attached to the 6-position of the nonreducing terminal Glc residue of DH-DG. DH-DG in PP and TH comprised 65.5% and 81.6% of the total glycolipids, respectively, and the ratios of PG and DH-DG to CL were 1.9 and 1.4 for PP, and 2.0 and 1.7 for TH. TLC immunostaining with human sera revealed antibodies for Galα1-2Glcα1-3′DG (LacDH-DG) from Lactobacillus species and Glcβ1-6Glcβ1-3′DG (StaDH-DG) from Staphylococcus species, but not for Glcα1-2Glcα1-3′DG (StrDH-DG) from Streptococcus species, in 2 out of 20 human sera. Given that one serum sample with anti-StaDH-DG antibodies was from a patient who had suffered food poisoning due to Staphylococcus aureus 6 months previously, the antibodies for bacterial DH-DG were thought to have arisen via bacterial infection. bacterial dihexaosyl diglyceride kojibiose bacteria for fermented food symbiotic bacteria anti-glycolipid antibody TLC immunostaining Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Bacterial glycosyl diglycerides, in which hydrophilic carbohydrates are linked with hydrophobic diglycerides (DG), are present in Gram-positive bacteria (but not in Gram-negative ones) at concentrations equivalent to those of phospholipids, with phosphatidyl glycerol (PG) and cardiolipin (CL) being the major membrane constituents [ 2 ]. The molecular species of DG moieties of PG, CL, and glycosyl DG in symbiotic bacteria living in the human oral cavity, intestine, and skin exhibit characteristic profiles containing unsaturated, cyclopropane ring-containing, and anteiso-type fatty acids, respectively, which provide the most suitable structures for bacteria to survive in the completely different environmental conditions that prevail at these anatomical sites. For example, in Streptococcus salivarius (SS) in the oral cavity, approximately 70% of the total molecular species were found to contain oleic (18:1) and palmitoleic (16:1) acids, which is suggested to be an adaptation to the particular temperature in the oral cavity. Meanwhile, half of the molecular species in Lactobacillus johnsonii (LJ) in the intestine contained 11,12-methylene-octadecanoic (cyclopropane ring-containing) acids (cy19:0), probably to avoid cleavage of the double bond of 18:1 upon exposure to digestive juice. In addition, 94% of the molecular species of DG moieties of PG, CL and glycosyl DG in Staphylococcus epidermidis (SE) in the skin were found to contain anteiso fatty acids (ai15:0 and ai17:0), which is suggested to be an adaptation providing a relatively solid biomembrane enabling survival in the arid environment of the skin [ 2 ]. As for the carbohydrate moieties of glycosyl DG, dihexaose (DH) is linked in a manner dependent on the specific bacterial species [i.e., Galα1-2Glcα1-3′DG (LacDH-DG) in Lactobacillus species, Glcα1-2Glcα1-3′DG (StrDH-DG, kojibiosyl DG) in Streptococcus species, and Glcβ1-6Glcβ1-3′DG (StaDH-DG, gentibiosyl DG) in Staphylococcus species] [ 3 – 5 ]. The essential function of DH-DG is to anchor teichoic acid through the carbohydrate moieties to retain thick glycopeptides in the cell wall of Gram-positive bacteria [ 3 ]. Therefore, bacterial glycolipids in the plasma membrane are thought to be hidden behind the cell wall. However, upon direct immunization of rabbits with LJ, SS, and SE, the antisera were found to react with the carbohydrate moieties of glycolipids in a manner dependent on the particular bacterial species. Specifically, although TLC immunostaining showed that anti-SE antisera preferentially reacted with StaDH-DG, anti-SS antisera reacted with StrDH-DG and, to a lesser extent, with LacDH-DG and StaDH-DG [ 2 ]. Notably, anti-LJ antiserum reacted intensely with Galα1-6LacDH-DG ( Lactobacillus trihexaosyl DG, LacTH-DG) and Galα1-6Galα1-6LacDH-DG ( Lactobacillus tetrahexaosyl DG, LacTetH-DG), as well as with LacDH-DG. In addition, the natural antibodies for LacTH-DG and LacTetH-DG found in 75% of human sera exhibited similar binding intensity to glycolipids of blood groups A and B, irrespective of the ABO blood group, indicating that bacterial species-specific glycolipids, even those present within the cell wall, possess immunological activity [ 2 , 4 ]. Moreover, the predominant Lactobacillus species in the digestive tract of immunodeficient scid mice was reported to be distinct from that in control mice under the same breeding conditions (i.e., L. murinus with Glcβ1-6LacDH-DG in scid and LJ with LacTetH-DG in control mice). This suggested that immune recognition through bacterial glycolipids is partly involved in the establishment of a symbiotic relationship between bacteria and host. The human immune system is continually exposed to exogenous bacteria through food, but the potential contribution of bacterial glycolipids to immune recognition in the intestinal tract to allow the absorption of food or to reject and expel it remains obscure. In terms of characterization of the glycolipids in bacteria used for the production of fermented foods, those in Lactobacillus species have been identified [ 4 ], but those in Pediococcus pentosaceus used to produce fermented soybean paste (miso) and Tetragenococcus halophilus used to produce soy sauce as traditional Japanese foods remain unknown. Accordingly, we determined the structures of bacterial glycolipids in Pediococcus and Tetragenococcus species in comparison with those in symbiotic bacteria, such as Lactobacillus , Staphylococcus , and Streptococcus species, to provide clues on why both of the former bacteria became important in the Japanese diet. Materials and methods Bacteria P. pentosaceus (PP, JCM 5885), T. halophilus (TH, JCM 5888), LJ (JCM1022), SE (JCM2414), and SS (JCM5707) were purchased from the Japan Collection of Microorganisms (JCM), RIKEN BioResource Center (Wako, Saitama, Japan). The culture media for bacteria were as follows: MRS (deMan, Rogosa, and Sharpe) broth (Beckton-Dickinson, Sparks, MD, USA) for LJ, GAM (Gifu Anaerobic Medium) broth (Nissui Pharma. Co., Tokyo) for PP, tryptic soy broth (Beckton-Dickinson) for SE and TH, and heart infusion broth (Beckton-Dickinson) for SS. The bacteria were first cultured on an agar plate (1.2% agar in the respective media), and the living ones were picked up from the colonies formed with a toothpick and cultured in liquid media. For lipid analysis, the bacteria were cultured in 500 mL of the respective liquid media at 32°C for 24 h, collected by centrifugation at 6,000 rpm for 20 min, washed with phosphate-buffered saline (PBS), and lyophilized. Materials Glycolipids, GlcCer, GalCer, LacCer, Glcα1-3′DG, Glcβ1-3′DG, LacDH-DG, StaDH-DG, StrDH-DG, and IV 3 GalNAcα-Gb 4 Cer (Forssman glycolipids), were purified in our laboratory [ 3 , 4 ]. Phosphatidylethanolamine (PE), PG, CL, and peroxidase-conjugated anti-human IgGAM antibodies were purchased from Sigma (St. Louis, MO, USA). The concentrations of standard phospholipids in chloroform/methanol (1:1, by vol.) were determined by the phosphomolybdate procedure after decomposition of the lipids with 70% HClO 4 and H 2 O 2 [ 5 ]. Normal human sera, blood groups A, B, AB, and O, were obtained from Keio University Hospital, and used in accordance with the guidelines of the hospital’s Committee for Informed Consent. Quantitation of bacterial lipids The lipid extracts derived from the lyophilized bacteria were partitioned by Folch’s procedure [ 4 ], and then aliquots corresponding to a dry weight of 0.5–1.0 mg were separated on glass-coated (Merck, Darmstadt, Germany) and plastic-coated TLC plates (Macherey-Nagel, Düren, Germany) with chloroform/methanol/water (65:25:4. by vol.). Detection was performed with cupric acetate–phosphoric acid reagent for all lipids, Dittmer’s reagent for phospholipids, ninhydrin reagent for aminolipids, and orcinol–sulfuric acid reagent for glycolipids, and by TLC immunostaining with human sera (1:100). For the quantitative determination of phospholipids and glycolipids, standard lipids [i.e., PG, CL, and N-stearoyl derivatives of GalCer and LacCer, and Glcα1-3′DG and StrDH-DG from SS (0.1–1.5 µg)], were developed on the same TLC plates. After visualization of phospholipids with cupric acetate–phosphoric acid and of glycolipids with orcinol–sulfuric acid, the density of spots was determined by image analysis (NIH image) for the preparation of standard curves [ 5 ]. Enzyme-linked immunosorbent assay (ELISA) LacDH-DG, StaDH-DG, StrDH-DG, and Forssman glycolipids were dissolved in ethanol (1 µg/50 µl) by sonication, and then put in individual wells of a microtiter plate (Thermo Fisher Scientific, Waltham, MA, USA), which was left at room temperature until the ethanol had completely evaporated off. The plate was blocked with 100 µl of bovine serum albumin (BSA, 1%) in PBS at 4ºC overnight, and then 100 µl of human serum diluted 1:100 with 1% BSA in PBS was added to the plate, followed by reaction at room temperature for 2 h. After washing the plate with 0.1% Tween 20 in PBS five times, the antibodies bound on the plate were detected by reaction with 100 µl of peroxidase-conjugated anti-human IgGAM antibodies diluted 1:2,000 with 1% BSA in PBS at room temperature for 2 h, followed by reaction with o-phenylenediamine (4.6 mM) and H 2 O 2 (0.015%) in 25 mM citrate-phosphate buffer (pH 5.0) as the substrates for peroxidase at room temperature for 15 min. The reaction was terminated by the addition of 4 M sulfuric acid (20 µl), and then the optical density at 490 nm was determined. The background values obtained on reaction with human sera in wells without glycolipids under the same conditions were subtracted from the values obtained as described above. Purification of glycolipids and phospholipids Total lipids were extracted from the lyophilized bacteria with chloroform/methanol/water (20:10:1, 10:20:1, and 1:1:0, by vol.), and then fractionated into neutral and acidic lipid fractions by DEAE-Sephadex (A-25, acetate form; GE Healthcare Bioscience, Piscataway, NJ, USA) column chromatography. After Folch partitioning, individual glycolipids and phospholipids were purified from each fraction using a silica gel (Iatrobeads 6RS8060; Iatron Laboratory, Tokyo, Japan) column by gradient elution with chloroform/methanol/water (85:15:0.2, 70:30:4, and 10:90:4, by vol.). The purity of purified lipids was examined by TLC as described above. Structural analysis of glycolipids and phospholipids The purified glycolipids and phospholipids were analyzed by positive- and negative-ion FAB-MS (JMS-700TKM; JEOL, Tokyo, Japan), respectively, with triethanolamine (TEA) as the matrix, and by proton magnetic resonance spectroscopy (JNM-ECP700, JEOL) with dimethyl sulfoxide-d6/D 2 O (98:2, by vol.). For determination of the fatty acid and carbohydrate compositions, they were methanolized with 5% HCl in methanol at 80°C for 16 h. The resulting fatty acid methyl esters were extracted with n-hexane, and 1-O-methyl hexoses in the methanol phase were converted into trimethylsilyl derivatives with pyridine/hexamethyl disilazane/trimethylchlorosilane (10:2:1, by vol.) at 60°C for 5 min. The fatty acid methyl esters and 1-O-methyl 2,3,4,6-tetra-O-trimethylsilyl hexoses were analyzed by GC-MS (GP5050; Shimadzu, Kyoto, Japan) equipped with a DB-1 column (0.25 mmø × 30 m) with temperature elevation from 150 to 250°C at 10°C/min. In addition, linkage analysis of carbohydrates was carried out by conversion of glycolipids to partially methylated aldohexitol acetates, followed by analysis using GC-MS with a DB-1 column with temperature elevation from 150 to 210°C at 4°C/min [ 2 , 4 ]. In addition, for determination of the carbohydrate sequences and anomeric configurations, glycolipids (2 µg) together with 100 µg/µl sodium taurocholate were hydrolyzed by incubation with α-glucosidase (rice; Sigma) in 50 mM citrate buffer (pH 4.5) at 37ºC for 4 h in a final volume of 100 µl. The obtained products were recovered by solvent partitioning with 100 µl of chloroform and 50 µl of methanol, followed by that with 100 µl of methanol/water (1:1, by vol.). Then, the samples were examined by TLC with chloroform/methanol/water (65:25:4, by vol.) and orcinol–sulfuric acid reagent for glycolipids, and with n-hexane/diethyl ether/acetic acid (80:30:4, by vol.) and cupric acetate–phosphoric acid reagent for DG [ 4 ]. Results P. pentosaseus and T. halophilus Both PP and TH produce lactic acid as the major metabolic end product of carbohydrate fermentation, and form an acidic environment to support yeast under conditions with rather high salt concentration during the processes of miso and soy sauce production [7]. Although the colonies of LJ, SE, and SS on agar plates in this study were spherical in shape, those of PP and TH were lens-shaped (Fig. 1). After picking up the bacteria from the colonies with a toothpick, culturing them in 500 mL of the respective liquid medium at 32 ºC for 24 h, and lyophilizing them, PP and TH were obtained at dried weights of 420±30 and 650±70 mg, respectively. Phospholipids in bacteria As shown in Fig. 2, the major phospholipids in PP and TH were CL and PG, as determined by comparison with the mobilities of their standards. Specifically, the migration of the standards closely resembled that of the samples on a glass plate, but CL on a plastic plate moved to the solvent front, and was separated from PG [2]. Their structures were also confirmed by negative-ion FABMS spectrometry, which gave molecular ions of [M-H] - for PG and [M-2H] - for CL [2]. On the basis of the fatty acid compositions of PG and CL as the methyl esters after methanolysis as determined by GC-MS, individual molecular species of PG and CL as the combinations of the fatty acids in their DG moieties were determined from their molecular ions, as shown in Table 1. The major molecular species of PG and CL in PP consisted of 18:1 and 19:0, and 18:1, 18:1, 19:0, and 19:0, while those in TH were 16:0 and 18:1, and 16:0, 18:0, 18:1, and 18:1, respectively. Glycolipids in bacteria As shown in Fig. 2B, three glycolipids were detected in both PP and TH on a TLC plate using orcinol–H 2 SO 4 reagent. The first and second migrating bands in PP and TH were neutral in nature and their mobilities were similar to those of MH-DG and DH-DG from SS, respectively. The third migrating bands in both PP and TH were fractionated into the acidic fractions by DEAE Sephadex column chromatography, and were positive with Dittmer’s reagent, suggestive of a DH-DG structure modified with phosphorus (designated as PO 3 -DH-DG in Fig. 2B). Upon GC-MS analysis of carbohydrate in the form of 1-O-methyl 2,3,4,6-tetra-O-trimethylsilyl derivatives after methanolysis, glucose was found to be the only carbohydrate in all glycolipids from PP and TH. As shown in Fig. 3 and Table 2, partially methylated aldohexitol acetates prepared from DH-DG in PP gave two peaks, whose retention times and mass spectra were identical to those of 1,5-di-O-acetyl 2,3,4,6-tetra-O-methyl Glc (terminal Glc) for peak a and 1,2,5-tri-O-acetyl 3,4,6-tri-O-methyl Glc (2-linked Glc) for peak b. In addition, 1 H-NMR spectra (Fig. 3C) contained doublet signals at d=4.913 ppm (J=3.2 Hz) and d=4.821 ppm (J=4.0 Hz), indicating that the anomers of both of the two Glc residues in DH-DG in PP were in the a-configuration. Accordingly, the structure of DH-DG from PP was shown to be Glca1-2Glca1-3¢DG, namely, kojibiosyl DG. Identical results were obtained for DH-DG from TH, whose structure was also determined to be Glca1-2Glca1-3¢DG (Table 2). Thus, the carbohydrate structures of DH-DG in Pediococcus and Tetragenococcus species were shown to be the same as those in Streptococcus species (StrDH-DG). The positive-ion FABMS spectra gave the abundant molecular ions [DH-DG+Na] + , m/z 967 (74.9%) for 18:1 and 18:1 from DH-DG of PP, and m/z 941 (44.6%) for 16:0 and 18:1 from DH-DG of TH (Fig. 4, Table 3). The glucose residues of MH-DG in both bacteria (Table 2) were susceptible to a-glucosidase (rice) to yield DG, although the a-1,2-linkage of the terminal Glc in DH-DG was not cleaved by a-glucosidase, which reacted with a1,4- and a1,6-linkages of Glc in oligosaccharides. Thus, MH-DGs in both PP and TH were suggested to be Glca1-3¢DG, whose molecular species were similar to those of DH-DG (Table 3). Meanwhile, 2-linked and 6-linked Glcs were detected from both PO 3 -DH-DG of PP and TH at a ratio of 1:1, indicating a modification of the 6-position of the terminal Glc of DH-DG (Table 2). The negative-ion FABMS spectra gave molecular ions of [M-H] - , m/z 1097, from PO 3 -DH-DG (18:1,18:1) of PP and m/z 1071 from PO 3 -DH-DG (16:0,18:1) of TH, suggesting that glycerol phosphate (Gro-P, molecular weight of 155) is attached to the 6-position of terminal Glc (Fig. 4, Table 3). The structures of PO 3 -DH-DG of both PP and TH were suggested to be 6-(Gro-PO 3 )-Glca1-2Glca1-3¢DG, which was already reported as a typical modified structure of kojibiosyl DG in Gram-positive bacteria [8,9]. Amounts of phospholipids and glycolipids in PP and TH On the basis of the data obtained by TLC densitometry, the amounts of CL, PG, and glycolipids in PP and TH were estimated, as shown in Table 4. The major phospholipids, CL and PG, amounted to 2.0 mg/mg dry weight for PP and 2.75 mg/mg dry weight for TH and the ratios of CL to PG were approximately 1:2 for both bacteria. The total amounts of glycolipids were 1.45 mg/mg dry weight for PP and 1.9 mg/mg dry weight for TH, of which Glca1-2Glca1-3¢DG (DH-DG) comprised 65.5% and 81.6% of the total, respectively. Thus, the lipid bilayers of both PP and TH consisted of CL, PG, and DH-DG. Antibodies for bacterial DH-DG in human sera Human sera of blood groups A, B, AB, and O were reacted with LacDH-DG, StaDH-DG, StrDH-DG, and Forssman glycolipids on an ELISA plate, and those giving optical densities in excess of 0.4 were able to generate positive bands for the respective glycolipids by TLC immunostaining (Table 5). The antibodies toward Forssman antigens were frequently detected in human sera (in 16 out of 20 sera), irrespective of the ABO blood group. As shown in Fig. 5A, high-performance TLC (HPTLC) revealed that the mobility of DH-DG from PP was identical to that of StrDH-DG from SS, but that from TH was slightly lower than that of StrDH-DG from SS, owing to the higher amount of palmitic acid in its DG moiety from TH. In addition, StaDH-DG with ai15:0 and ai17:0 from SE migrated to a position lower than DH-DG from TH. Meanwhile, all DH-DGs on the plastic TLC plate had the same mobility (Fig. 5B). Serum samples 3 and 13, and Serum samples 5 and 13, were found to contain antibodies for LacDH-DG and StaDH-DG, respectively (Fig. 5B). In particular, since serum 13 was from a patient who had suffered fromfood poisoning due to Staphylococcus aureus 6 months previously, antibodies for StaDH-DG (gentibiosyl DG) were thought to have been generated by staphylococcal infection. Moreover, the optical densities of antibody binding to StrDH-DG of all sera examined by ELISA were lower than 0.17, and the binding of antibodies to StrDH-DG was not detected by TLC immunostaining. Discussion Upon comparing the glycolipid structures of symbiotic bacteria living in the human oral cavity, intestine, and skin, they were each shown to have characteristic fatty acid compositions of their DG moieties, as potential adaptations to the completely different environmental conditions that prevail at these anatomical locations. Meanwhile, those from PP and TH were composed of unsaturated fatty acids as more than 70% of the total molecular species, resembling the findings for SS in the oral cavity. Moreover, the carbohydrate structures of DH-DGs were characteristic of the particular bacterial species, providing a landmark for the bacterial species to distinguish each other and probably for their hosts to recognize them. The disaccharide structures of DH-DGs in Pediococcus and Tetragenococcus species were kojibiose, in which two α-glucoses are linked by an α1–2 linkage, and were also identical to StrDH-DG in SS in the oral cavity. Since α-glucose is a constituent of glycogen and starch as the major nutrients in humans, the intestinal immune system is continuously exposed to it to ensure tolerance to it. This could help to explain why SS can inhabit the oral cavity as a symbiotic bacterium, and why PP and TH were selected as bacteria for the preparation of fermented foods, namely, miso and soy sauce, respectively. In addition, although α1–4 and α1–6 linkages of Glc in glycogen and starch are readily hydrolyzed by α-glucosidase, the α1–2 linkage is resistant to enzymatic cleavage, so kojibiose is used as an inhibitor of α-glucosidase [ 10 , 11 ]. Thus, the glycosidic linkage in StrDH-DG in SS, PP, and TH might not be hydrolyzed during the digestion process, despite bacterial mortality due to the strongly acidic conditions in the stomach, although monoglucosyl DG was reportedly hydrolyzed to yield Glc and DG by α-glucosidase [ 4 ]. In contrast, StrDH-DG, as a derivative of kojibiose linked with DG, did not inhibit the activity of α-glucosidase (rice) with p -nitrophenyl α-D-glucopyranoside as the substrate. This showed that the enzymatic breakdown of starch and glycogen by α-glucosidase is not affected by StrDH-DG in SS, PP, and TH (Iwamori et al., unpublished observation). Similarly, the terminal carbohydrate linkages of DH-DGs in LJ and SE (i.e., Galα1-2 of LacDH-DG and Glcβ1-6 of StaDH-DG) were thought to be resistant to digestion because of the absence of exoglycosidases for them in the digestive tract. In fact, LacTetH-DG, LacTH-DG, and LacDH-DG in LJ were reportedly maintained without degradation in the cecal and colonic contents and feces of mice [ 12 ]. In the case of LJ, Gg 4 Cer in the duodenal, jejunal, and ileal epithelia of the murine digestive tract was reported as a receptor enabling the formation of intestinal bacterial flora [ 13 , 14 ], and was also excreted into the feces without degradation. This indicates that Gg 4 Cer in the feces might facilitate the discharge of LJ and other bacteria using it as a receptor, by attaching to them to prevent their irregular diffusion upon their release from intestinal epithelia into the digestive tract [ 12 ]. In fact, upon TLC immunostaining with anti-LJ, anti-SE, and anti-SS antisera of lipid extracts from the feces of 2-month-old female mice, although several bands appeared in the regions of MH-DG and DH-DG and could not be distinguished among them, LacTH-DG and LacTetH-DG were only detected with anti-LJ antisera, at amounts corresponding to approximately 1.4 × 10 8 cells/g dry weight [ 11 ]. In addition, Gg 4 Cer, corresponding to approximately 20% of the total amounts of Gg 4 Cer in the duodenal, jejunal, and ileal epithelia, was excreted into the feces without degradation [ 12 ]. Glycolipids are generally known to be strongly antigenic compounds, and antibodies against their carbohydrate moieties are readily generated by epidermal immunization with tissues and cells, as well as with purified glycolipids [ 14 , 15 ]. Therefore, bacterial glycolipids in the digestive tract are expected to be recognized as foreign antigens to yield circulating antibodies in sera through their processing by gut-associated lymphoid tissues in Peyer’s patches [ 16 , 17 ]. Among glycolipids in LJ, SE, and SS, those with three or four carbohydrates, namely, LacTH-DG and LacTetH-DG, generated antibodies at greater intensity and higher specificity than those with one or two carbohydrates, upon direct immunization of rabbits with bacteria together with Freund’s complete adjuvant [ 12 ]. In practice, antibodies against LacTH-DG and LacTetH-DG in LJ were detected in human sera at frequencies and intensities similar to those against ABO-blood group-related glycolipids [ 4 , 6 , 18 ]. Since LacTH-DG and LacTetH-DG are contained in various Lactobacillus species and strains, continuous stimulation of the gut-associated lymphoid tissues by intestinal symbiotic bacteria and bacteria in fermented foods might result in the production of antibodies for them. However, it has remained unknown whether such antibodies play a role in preventing bacteria from invading the bloodstream. Interestingly, anti-glycolipid antibodies generated upon infection with Campylobacter jejuni are known to be closely associated with the onset of autoimmune diseases, such as Guillain–Barré and Miller–Fisher syndromes [ 19 – 21 ]. In the case of C. jejuni as a Gram-negative bacterium, the terminal structures of lipooligosaccharides were identical to those of glycosphingolipids in human neural tissues [ 19 ], resulting in the production of antibodies that cross-react with glycosphingolipids, followed by antibody-mediated inflammatory neuropathies [ 20 , 21 ]. However, since structures mimicking Forssman and ABO-blood group-related oligosaccharides have yet to be detected in any bacteria, the original stimuli generating the antibodies against them are obscure, although several hypotheses explaining the production of natural antibodies have been proposed [ 22 , 23 ]. In contrast, antibodies against DH-DG, with a rather short carbohydrate chain, were thought to be generated by abnormal stimulation of the respective bacteria. As shown in this paper, anti-StaDH-DG antibodies were detected in 2 out of 20 human sera, one of which was from a patient who had suffered from food poisoning due to S. aureus 6 months previously. It is likely that, although S. epidermidis as a symbiotic bacterium normally stimulates the immune system in the skin on a daily basis, abnormal stimulation by S. aureus might cause the production of antibodies against StaDH-DG (gentibiosyl DG). To test this hypothesis, there is a need for further examinations with a large number of human serum samples in order to clarify the correlation between anti-DH-DG antibodies and disorders of the intestinal microbiota. In addition, α-galactosyl ceramide from marine sponge and α-galacturonyl ceramide from Sphingomonas bacteria were revealed to exert immunoregulatory activity on CD1d-restricted natural killer T cells [ 24 – 26 ]. It is likely that bacterial glycolipids possessing similar bioactivity are found in the Gram-positive bacteria inhabiting various anatomical sites of the human body. A systematic analysis of glycolipid structures in various bacterial species is required to understand glycolipid signaling between bacteria and immunocytes. Abbreviations The nomenclature for glycosphingolipids is based on the recommendations of the IUPAC IUB Commission on Biochemical Nomenclature [1] DG, diglyceride; Gro, glycerol; LacDH DG, Lactobacillus dihexaosyl diglyceride, Galα1-2Glcα1-3′DG; StrDH-DG, Streptococcus dihexaosyl diglyceride, Glcα1-2Glcα1-3′DG; StaDH-DG, Staphylococcus dihexaosyl diglyceride, Glcβ1-6Glcβ1-3′DG; PG, phosphatidyl glycerol; CL, cardiolipin; TLC, thin layer chromatography Declarations Compliance with ethical standards Conflict of interest Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Funding support: no Author Contribution Both authors equally contributed for this study. References IUPAC-IUB Commission on Biochemical Nomenclature: The Nomenclature of Lipids. Eur. J. 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Biophys. Acta 575 , 389-398 (1979) Laparra, J.M., Diez-Municio, M., Moreno, F.J., Herreno, M.: Kojibiose ameliorates arachidic acid-induced metabolic alterations in hyperglycaemic rats. Br. J. Nutr. 114 , 1395-1402 (2015) Forsgard, R.A., Lactose digestion in humans: intestinal lactose appears to be constitutive whereas the colonic microbiome is adaptable. Am. J. Clin. Nutr. 110 , 273-279 (2019) Iwamori, M., Iwamori, Y., Adachi, S., Nomura, T. Excretion into faeces of asialoGM1 in the murine defective tract and Lactobacillus johnsoni exhibiting binding ability toward asialoGM1. A possible role of epithelial glycolipids in the discharge of intestinal bacteria. Glycoconj. J. 28 , 21-30 (2011) Karlsson, K.A. Animal glycoshingolipids as membrane attachment sites for bacteria. Ann. Rev. Biochem. 58 , 309-350 (1989) Iwamori, M. A new turning point in glycosphingolipid research. Human Cell 18 , 117-133 (2005) Iwamori, M., Murata, M., Toyoda, M., Iwamori, Y.: Contribution of glycolipids to species-specific antigens on erythrocytes of several animal species as to recognition of antigens with rabbit anti-glycolipids and anti-erythrocyte antisera. Glycoconj. J. 26 , 467-476 (2009) Tuganbaev, T., Honda, K.: Non-zero-sum microbiome immune system interactions. Eur. J. Immunol. 51 ,2120-2136 (2021) Artis,D., Spits,H. The biology of innate lymphoid cells. Nature 517, 293-301 (2015) Tanaka, K., Suzuki, A., Aoki, D., Iwamori, M.: Characterization of a novel glycolipid with a difucosylated H-antigen in human blood group O erythrocytes with monoclonal antibody HMMC and its detection in human uterine cervical carcinoma cells. Glcoconj.J. 36 , 219-226 (2019) Godschalk, P.C., Kuijf, M.L., Li, J., St. Michael, F., Ang, C.W., Jacobs, B.C., Karwaski, M.F., Brochu, D., Moterassed, A., Endtz, H. P., van Belkum, A., Gilbert, M.: Structural characterization of Campylobacter Jejuni lipooligosaccharidre outer cores associated with Guillain-Barre and Miller-Fisher syndromes. Infect. Immun. 75 , 1245-1254 (2007) Wakerley, B. R., Uncini, A., Yuki, N., Guillain-Barre and Miller-Fisher syndromes-new diagnostic classicication. Nat. Rev. Neurol. 10 , 537-544 (2014) Martin-Anguilar, L., Pascual-Goni, E., Querd, L.: Autotntibodies in immune-mediated inflammatory neuropathies. Med.Clin. 153 , 360-367 (2019) Arthur, C. M., Stowell, S. R.: The development and consequences of red blood cell alloimmunization. Annu. Rev. Pathol. 18 , 537-564 (2023) Avci, F.Y., Kasper, D.L.: How bacterial carbohydrate influence the adaptive immune system. Annu. Rev. Immunol. 28 , 107-130 (2010) Nishimura, T., Kitamura, H., Iwakabe, K., Yahata, T., Ohta, A., Sato, M., Takeda, K., Okumura, K., Vankaer, L., Kawano, T., Taniguchi, M., Nakui, M., Sekimoto, M., Koga, T.: The interface between innate and acquired immunity: glycolipid antigen presentation by CD1d-expressing dendritic cells to NKT cells induce the differentiation of antigen-specific cytotoxic T lymphocytes. Int. Immunol. 12 , 987-994 (2000) Wu, D., Xing, G., Poles, M.A., Wong, C.: Bacterial glycolipids and analogs as antigens for CD1d-restricted NKT cells. Proc. Natl. Acad. Sci. USA 102 , 1351-1356 (2005) Abdel-Mawgoud, A. M.: Stephanopoulos,G., Simple glycolipids of microbes: Chemistry, biological activity and metabolic engineering. Synth. Syst. Biotechnol. 15 , 3-19 (2017) Tables Tables 1-5 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Table2.docx Table3.docx Table4.docx Table5.docx Cite Share Download PDF Status: Published Journal Publication published 15 Jul, 2025 Read the published version in Glycoconjugate Journal → Version 1 posted Editorial decision: Revision requested 13 Jun, 2025 Reviews received at journal 11 Jun, 2025 Reviewers agreed at journal 09 Jun, 2025 Reviews received at journal 05 Apr, 2025 Reviewers agreed at journal 27 Mar, 2025 Reviewers invited by journal 27 Mar, 2025 Editor assigned by journal 27 Mar, 2025 Submission checks completed at journal 27 Mar, 2025 First submitted to journal 19 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6265412","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":439052145,"identity":"ffc8c326-e18f-4b49-84fa-7354eb3aae80","order_by":0,"name":"Masao Iwamori","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYBACNgglwSDBDCINbEAkBDA2EKcljbAWOJCA4MMILbgAn0Ty0w0f91gwSLbzHrzxo+B8Yv/s5oMPGGpsGJhnY7eGTSLN7OaMZxIM0sx8yZY9BrcTZ9w5lmzAcCyNgXHOAexapBPMbvMckGCQY+Yxk+ABamm4kWMmwdhwmIFxRgIOLenf4Fok/xicS5xPWEsOxBZpoBZpHoMDiRsIapF/U3ZzBlCLZDOPsbWMQbLxxhtpyQYJx9J4cPlFvuf4thsfDtQxSJw/Y3jzzR872Xk3kg8++FBjI2eII8RgoB4m7QhmAJ3EYzgDrw4EsEc4gFCcjoJRMApGwUgBAO2TWJ087x0oAAAAAElFTkSuQmCC","orcid":"","institution":"Toho University","correspondingAuthor":true,"prefix":"","firstName":"Masao","middleName":"","lastName":"Iwamori","suffix":""},{"id":439052146,"identity":"2010818e-5b27-4e72-ba33-68d1ea824cbc","order_by":1,"name":"Kyoko Tanaka","email":"","orcid":"","institution":"Toho University","correspondingAuthor":false,"prefix":"","firstName":"Kyoko","middleName":"","lastName":"Tanaka","suffix":""}],"badges":[],"createdAt":"2025-03-20 02:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6265412/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6265412/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10719-025-10190-1","type":"published","date":"2025-07-15T16:05:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80710393,"identity":"1f2dab46-f049-43b1-82a3-8a6a9aa571df","added_by":"auto","created_at":"2025-04-16 09:00:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28116,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial colonies of\u003cem\u003e P. pentosaceus\u003c/em\u003e (PP) and\u003cem\u003e T. halophilus\u003c/em\u003e (TH). PP and TH were cultured in GAM and tryptic soy broth containing 1.2% agar at 32 ℃ for 24 h, respectively. The mean diameters of the major axis were 3 mm for PP and 5 mm for TH.\u003c/p\u003e","description":"","filename":"BacFig.11.png","url":"https://assets-eu.researchsquare.com/files/rs-6265412/v1/735dec1aef5904b86bf9e186.png"},{"id":80710391,"identity":"51f86eb2-7c2e-4459-82e3-709eaf98ac5c","added_by":"auto","created_at":"2025-04-16 09:00:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23158,"visible":true,"origin":"","legend":"\u003cp\u003eTLC of lipids from TH and PP. The total lipids extracted from TH and PP, corresponding to a dry weight of 0.5 mg, were developed with chloroform/methanol/water (65:25:4, by vol.) and the spots were detected with cupric acetate-phosphoric acid (A) and orcinol-sulfuric acid (B), respectively.\u003c/p\u003e","description":"","filename":"BacFig.22.png","url":"https://assets-eu.researchsquare.com/files/rs-6265412/v1/ecb72016e810e0d872914be7.png"},{"id":80710399,"identity":"6756c2ad-fbd1-4239-9ffa-6d8e54514cb9","added_by":"auto","created_at":"2025-04-16 09:00:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":47871,"visible":true,"origin":"","legend":"\u003cp\u003eGC-MS chromatogram (A) and mass spectra (B) of partially methylated aldohexitol acetates prepared from DH-DG of PP, and NMR spectrum of DH-DG of PP (C). Mass spectra a and b in Fig. 3B correspond to peaks a and b in Fig. 3A, which were characterized as terminal Glc and 2-linked Glc, respectively, while peaks a and b in Fig. 3C were characterized as terminal Glc and internal Glc (2-linked Glc), respectively.\u003c/p\u003e","description":"","filename":"BacFig.31.png","url":"https://assets-eu.researchsquare.com/files/rs-6265412/v1/ee0f0197424261be8ad430d5.png"},{"id":80710395,"identity":"874b1b34-04fd-4c48-bd7b-9884306baee7","added_by":"auto","created_at":"2025-04-16 09:00:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20829,"visible":true,"origin":"","legend":"\u003cp\u003ePositive-ion FABMS spectra of DH-DG (A and B) and negative-ion FABMS spectra of PO\u003csub\u003e3\u003c/sub\u003e-DH-DG (C and D) from PP (A and C) and TH (B and D).\u003c/p\u003e","description":"","filename":"BacFig.41.png","url":"https://assets-eu.researchsquare.com/files/rs-6265412/v1/57d0af6788971fc9b908e5ed.png"},{"id":80711352,"identity":"dd0c216e-a001-4989-abc7-832ae15125c1","added_by":"auto","created_at":"2025-04-16 09:08:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":28775,"visible":true,"origin":"","legend":"\u003cp\u003eHPTLC (A) and TLC immunostaining with plastic-coated TLC plates (B) of DH-DGs.\u003c/p\u003e\n\u003cp\u003eLacDH-DG (1), StaDH-DG (2), StrDH-DG (3), and DH-DGs from PP (4) and from TH (5) were developed on HPTLC plates (A) and plastic-coated TLC plates (B) with chloroform/methanol/water (65:25:4, by vol.) and then detected with orcinol–H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e reagent (A) and by TLC immunostaining with human sera 3, 5, and 13 in Table 5.\u003c/p\u003e","description":"","filename":"BacFig.51.png","url":"https://assets-eu.researchsquare.com/files/rs-6265412/v1/fd3af75ac9afe08d9aca9fad.png"},{"id":87220355,"identity":"383947f4-30bc-443a-9cfa-dda335fac9de","added_by":"auto","created_at":"2025-07-21 16:11:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":627764,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6265412/v1/d7c31db0-e6f6-4974-9d67-b326c0040230.pdf"},{"id":80711349,"identity":"50ad1694-c350-441d-9694-d0135a29daf4","added_by":"auto","created_at":"2025-04-16 09:08:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19699,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6265412/v1/a71ec97555a410f97d69f08f.docx"},{"id":80711350,"identity":"a05bcf25-504b-4304-978b-c31078a0eddb","added_by":"auto","created_at":"2025-04-16 09:08:07","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17344,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6265412/v1/be888d9ac8f985bec4bc27ed.docx"},{"id":80711351,"identity":"4c08304f-f94a-436e-9e67-bc74a37bed52","added_by":"auto","created_at":"2025-04-16 09:08:07","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19478,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6265412/v1/c3c803718994ef3e341d5897.docx"},{"id":80710397,"identity":"2a552633-2d2c-4049-ab47-1ea100e01d4f","added_by":"auto","created_at":"2025-04-16 09:00:07","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17320,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.docx","url":"https://assets-eu.researchsquare.com/files/rs-6265412/v1/1d6427dcfc554a60411929e6.docx"},{"id":80710401,"identity":"9b6e5abf-b9c5-4c27-89eb-759569e45da0","added_by":"auto","created_at":"2025-04-16 09:00:07","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":19475,"visible":true,"origin":"","legend":"","description":"","filename":"Table5.docx","url":"https://assets-eu.researchsquare.com/files/rs-6265412/v1/91775102c070fa95b0a00ff8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bacterial glycolipids in Pediococcus pentosaceus for fermented soybean paste (miso) and Tetragenococcus halophilus for soy sauce","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBacterial glycosyl diglycerides, in which hydrophilic carbohydrates are linked with hydrophobic diglycerides (DG), are present in Gram-positive bacteria (but not in Gram-negative ones) at concentrations equivalent to those of phospholipids, with phosphatidyl glycerol (PG) and cardiolipin (CL) being the major membrane constituents [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The molecular species of DG moieties of PG, CL, and glycosyl DG in symbiotic bacteria living in the human oral cavity, intestine, and skin exhibit characteristic profiles containing unsaturated, cyclopropane ring-containing, and anteiso-type fatty acids, respectively, which provide the most suitable structures for bacteria to survive in the completely different environmental conditions that prevail at these anatomical sites. For example, in \u003cem\u003eStreptococcus salivarius\u003c/em\u003e (SS) in the oral cavity, approximately 70% of the total molecular species were found to contain oleic (18:1) and palmitoleic (16:1) acids, which is suggested to be an adaptation to the particular temperature in the oral cavity. Meanwhile, half of the molecular species in \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e (LJ) in the intestine contained 11,12-methylene-octadecanoic (cyclopropane ring-containing) acids (cy19:0), probably to avoid cleavage of the double bond of 18:1 upon exposure to digestive juice. In addition, 94% of the molecular species of DG moieties of PG, CL and glycosyl DG in \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e (SE) in the skin were found to contain anteiso fatty acids (ai15:0 and ai17:0), which is suggested to be an adaptation providing a relatively solid biomembrane enabling survival in the arid environment of the skin [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs for the carbohydrate moieties of glycosyl DG, dihexaose (DH) is linked in a manner dependent on the specific bacterial species [i.e., Galα1-2Glcα1-3\u0026prime;DG (LacDH-DG) in \u003cem\u003eLactobacillus\u003c/em\u003e species, Glcα1-2Glcα1-3\u0026prime;DG (StrDH-DG, kojibiosyl DG) in \u003cem\u003eStreptococcus\u003c/em\u003e species, and Glcβ1-6Glcβ1-3\u0026prime;DG (StaDH-DG, gentibiosyl DG) in \u003cem\u003eStaphylococcus\u003c/em\u003e species] [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The essential function of DH-DG is to anchor teichoic acid through the carbohydrate moieties to retain thick glycopeptides in the cell wall of Gram-positive bacteria [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, bacterial glycolipids in the plasma membrane are thought to be hidden behind the cell wall. However, upon direct immunization of rabbits with LJ, SS, and SE, the antisera were found to react with the carbohydrate moieties of glycolipids in a manner dependent on the particular bacterial species. Specifically, although TLC immunostaining showed that anti-SE antisera preferentially reacted with StaDH-DG, anti-SS antisera reacted with StrDH-DG and, to a lesser extent, with LacDH-DG and StaDH-DG [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Notably, anti-LJ antiserum reacted intensely with Galα1-6LacDH-DG (\u003cem\u003eLactobacillus\u003c/em\u003e trihexaosyl DG, LacTH-DG) and Galα1-6Galα1-6LacDH-DG (\u003cem\u003eLactobacillus\u003c/em\u003e tetrahexaosyl DG, LacTetH-DG), as well as with LacDH-DG. In addition, the natural antibodies for LacTH-DG and LacTetH-DG found in 75% of human sera exhibited similar binding intensity to glycolipids of blood groups A and B, irrespective of the ABO blood group, indicating that bacterial species-specific glycolipids, even those present within the cell wall, possess immunological activity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moreover, the predominant \u003cem\u003eLactobacillus\u003c/em\u003e species in the digestive tract of immunodeficient scid mice was reported to be distinct from that in control mice under the same breeding conditions (i.e., \u003cem\u003eL. murinus\u003c/em\u003e with Glcβ1-6LacDH-DG in scid and LJ with LacTetH-DG in control mice). This suggested that immune recognition through bacterial glycolipids is partly involved in the establishment of a symbiotic relationship between bacteria and host.\u003c/p\u003e \u003cp\u003eThe human immune system is continually exposed to exogenous bacteria through food, but the potential contribution of bacterial glycolipids to immune recognition in the intestinal tract to allow the absorption of food or to reject and expel it remains obscure. In terms of characterization of the glycolipids in bacteria used for the production of fermented foods, those in \u003cem\u003eLactobacillus\u003c/em\u003e species have been identified [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], but those in \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e used to produce fermented soybean paste (miso) and \u003cem\u003eTetragenococcus halophilus\u003c/em\u003e used to produce soy sauce as traditional Japanese foods remain unknown. Accordingly, we determined the structures of bacterial glycolipids in \u003cem\u003ePediococcus\u003c/em\u003e and \u003cem\u003eTetragenococcus\u003c/em\u003e species in comparison with those in symbiotic bacteria, such as \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e, and \u003cem\u003eStreptococcus\u003c/em\u003e species, to provide clues on why both of the former bacteria became important in the Japanese diet.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eBacteria\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. pentosaceus\u003c/em\u003e (PP, JCM 5885), \u003cem\u003eT. halophilus\u003c/em\u003e (TH, JCM 5888), LJ (JCM1022), SE (JCM2414), and SS (JCM5707) were purchased from the Japan Collection of Microorganisms (JCM), RIKEN BioResource Center (Wako, Saitama, Japan). The culture media for bacteria were as follows: MRS (deMan, Rogosa, and Sharpe) broth (Beckton-Dickinson, Sparks, MD, USA) for LJ, GAM (Gifu Anaerobic Medium) broth (Nissui Pharma. Co., Tokyo) for PP, tryptic soy broth (Beckton-Dickinson) for SE and TH, and heart infusion broth (Beckton-Dickinson) for SS. The bacteria were first cultured on an agar plate (1.2% agar in the respective media), and the living ones were picked up from the colonies formed with a toothpick and cultured in liquid media. For lipid analysis, the bacteria were cultured in 500 mL of the respective liquid media at 32\u0026deg;C for 24 h, collected by centrifugation at 6,000 rpm for 20 min, washed with phosphate-buffered saline (PBS), and lyophilized.\u003c/p\u003e \u003cp\u003eMaterials\u003c/p\u003e \u003cp\u003eGlycolipids, GlcCer, GalCer, LacCer, Glcα1-3\u0026prime;DG, Glcβ1-3\u0026prime;DG, LacDH-DG, StaDH-DG, StrDH-DG, and IV\u003csup\u003e3\u003c/sup\u003eGalNAcα-Gb\u003csub\u003e4\u003c/sub\u003eCer (Forssman glycolipids), were purified in our laboratory [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Phosphatidylethanolamine (PE), PG, CL, and peroxidase-conjugated anti-human IgGAM antibodies were purchased from Sigma (St. Louis, MO, USA). The concentrations of standard phospholipids in chloroform/methanol (1:1, by vol.) were determined by the phosphomolybdate procedure after decomposition of the lipids with 70% HClO\u003csub\u003e4\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Normal human sera, blood groups A, B, AB, and O, were obtained from Keio University Hospital, and used in accordance with the guidelines of the hospital\u0026rsquo;s Committee for Informed Consent.\u003c/p\u003e \u003cp\u003eQuantitation of bacterial lipids\u003c/p\u003e \u003cp\u003eThe lipid extracts derived from the lyophilized bacteria were partitioned by Folch\u0026rsquo;s procedure [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and then aliquots corresponding to a dry weight of 0.5\u0026ndash;1.0 mg were separated on glass-coated (Merck, Darmstadt, Germany) and plastic-coated TLC plates (Macherey-Nagel, D\u0026uuml;ren, Germany) with chloroform/methanol/water (65:25:4. by vol.). Detection was performed with cupric acetate\u0026ndash;phosphoric acid reagent for all lipids, Dittmer\u0026rsquo;s reagent for phospholipids, ninhydrin reagent for aminolipids, and orcinol\u0026ndash;sulfuric acid reagent for glycolipids, and by TLC immunostaining with human sera (1:100). For the quantitative determination of phospholipids and glycolipids, standard lipids [i.e., PG, CL, and N-stearoyl derivatives of GalCer and LacCer, and Glcα1-3\u0026prime;DG and StrDH-DG from SS (0.1\u0026ndash;1.5 \u0026micro;g)], were developed on the same TLC plates. After visualization of phospholipids with cupric acetate\u0026ndash;phosphoric acid and of glycolipids with orcinol\u0026ndash;sulfuric acid, the density of spots was determined by image analysis (NIH image) for the preparation of standard curves [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/p\u003e \u003cp\u003eLacDH-DG, StaDH-DG, StrDH-DG, and Forssman glycolipids were dissolved in ethanol (1 \u0026micro;g/50 \u0026micro;l) by sonication, and then put in individual wells of a microtiter plate (Thermo Fisher Scientific, Waltham, MA, USA), which was left at room temperature until the ethanol had completely evaporated off. The plate was blocked with 100 \u0026micro;l of bovine serum albumin (BSA, 1%) in PBS at 4\u0026ordm;C overnight, and then 100 \u0026micro;l of human serum diluted 1:100 with 1% BSA in PBS was added to the plate, followed by reaction at room temperature for 2 h. After washing the plate with 0.1% Tween 20 in PBS five times, the antibodies bound on the plate were detected by reaction with 100 \u0026micro;l of peroxidase-conjugated anti-human IgGAM antibodies diluted 1:2,000 with 1% BSA in PBS at room temperature for 2 h, followed by reaction with o-phenylenediamine (4.6 mM) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0.015%) in 25 mM citrate-phosphate buffer (pH 5.0) as the substrates for peroxidase at room temperature for 15 min. The reaction was terminated by the addition of 4 M sulfuric acid (20 \u0026micro;l), and then the optical density at 490 nm was determined. The background values obtained on reaction with human sera in wells without glycolipids under the same conditions were subtracted from the values obtained as described above.\u003c/p\u003e \u003cp\u003ePurification of glycolipids and phospholipids\u003c/p\u003e \u003cp\u003eTotal lipids were extracted from the lyophilized bacteria with chloroform/methanol/water (20:10:1, 10:20:1, and 1:1:0, by vol.), and then fractionated into neutral and acidic lipid fractions by DEAE-Sephadex (A-25, acetate form; GE Healthcare Bioscience, Piscataway, NJ, USA) column chromatography. After Folch partitioning, individual glycolipids and phospholipids were purified from each fraction using a silica gel (Iatrobeads 6RS8060; Iatron Laboratory, Tokyo, Japan) column by gradient elution with chloroform/methanol/water (85:15:0.2, 70:30:4, and 10:90:4, by vol.). The purity of purified lipids was examined by TLC as described above.\u003c/p\u003e \u003cp\u003eStructural analysis of glycolipids and phospholipids\u003c/p\u003e \u003cp\u003eThe purified glycolipids and phospholipids were analyzed by positive- and negative-ion FAB-MS (JMS-700TKM; JEOL, Tokyo, Japan), respectively, with triethanolamine (TEA) as the matrix, and by proton magnetic resonance spectroscopy (JNM-ECP700, JEOL) with dimethyl sulfoxide-d6/D\u003csub\u003e2\u003c/sub\u003eO (98:2, by vol.). For determination of the fatty acid and carbohydrate compositions, they were methanolized with 5% HCl in methanol at 80\u0026deg;C for 16 h. The resulting fatty acid methyl esters were extracted with n-hexane, and 1-O-methyl hexoses in the methanol phase were converted into trimethylsilyl derivatives with pyridine/hexamethyl disilazane/trimethylchlorosilane (10:2:1, by vol.) at 60\u0026deg;C for 5 min. The fatty acid methyl esters and 1-O-methyl 2,3,4,6-tetra-O-trimethylsilyl hexoses were analyzed by GC-MS (GP5050; Shimadzu, Kyoto, Japan) equipped with a DB-1 column (0.25 mm\u0026oslash; \u0026times; 30 m) with temperature elevation from 150 to 250\u0026deg;C at 10\u0026deg;C/min. In addition, linkage analysis of carbohydrates was carried out by conversion of glycolipids to partially methylated aldohexitol acetates, followed by analysis using GC-MS with a DB-1 column with temperature elevation from 150 to 210\u0026deg;C at 4\u0026deg;C/min [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, for determination of the carbohydrate sequences and anomeric configurations, glycolipids (2 \u0026micro;g) together with 100 \u0026micro;g/\u0026micro;l sodium taurocholate were hydrolyzed by incubation with α-glucosidase (rice; Sigma) in 50 mM citrate buffer (pH 4.5) at 37\u0026ordm;C for 4 h in a final volume of 100 \u0026micro;l. The obtained products were recovered by solvent partitioning with 100 \u0026micro;l of chloroform and 50 \u0026micro;l of methanol, followed by that with 100 \u0026micro;l of methanol/water (1:1, by vol.). Then, the samples were examined by TLC with chloroform/methanol/water (65:25:4, by vol.) and orcinol\u0026ndash;sulfuric acid reagent for glycolipids, and with n-hexane/diethyl ether/acetic acid (80:30:4, by vol.) and cupric acetate\u0026ndash;phosphoric acid reagent for DG [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eP. pentosaseus\u003c/em\u003e and\u0026nbsp;\u003cem\u003eT. halophilus\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBoth PP and TH produce lactic acid as the major metabolic end product of carbohydrate fermentation, and form an acidic environment to support yeast under conditions with rather high salt concentration during the processes of miso and soy sauce production [7]. Although the colonies of LJ, SE, and SS on agar plates in this study were spherical in shape, those of PP and TH were lens-shaped (Fig. 1). After picking up the bacteria from the colonies with a toothpick, culturing them in 500 mL of the respective liquid medium at 32 ºC for 24 h, and lyophilizing them, PP and TH were obtained at dried weights of 420±30 and 650±70 mg, respectively.\u003c/p\u003e\n\u003cp\u003ePhospholipids in bacteria\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 2, the major phospholipids in PP and TH were CL and PG, as determined by comparison with the mobilities of their standards. Specifically, the migration of the standards closely resembled that of the samples on a glass plate, but CL on a plastic plate moved to the solvent front, and was separated from PG [2]. Their structures were also confirmed by negative-ion FABMS spectrometry, which gave molecular ions of [M-H]\u003csup\u003e-\u003c/sup\u003e for PG and [M-2H]\u003csup\u003e-\u003c/sup\u003e for CL [2]. On the basis of the fatty acid compositions of PG and CL as the methyl esters after methanolysis as determined by GC-MS, individual molecular species of PG and CL as the combinations of the fatty acids in their DG moieties were determined from their molecular ions, as shown in Table 1. The major molecular species of PG and CL in PP consisted of 18:1 and 19:0, and 18:1, 18:1, 19:0, and 19:0, while those in TH were 16:0 and 18:1, and 16:0, 18:0, 18:1, and 18:1, respectively.\u003c/p\u003e\n\u003cp\u003eGlycolipids in bacteria\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 2B, three glycolipids were detected in both PP and TH on a TLC plate using orcinol–H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e reagent. The first and second migrating bands in PP and TH were neutral in nature and their mobilities were similar to those of MH-DG and DH-DG from SS, respectively. The third migrating bands in both PP and TH were fractionated into the acidic fractions by DEAE Sephadex column chromatography, and were positive with Dittmer’s reagent, suggestive of a DH-DG structure modified with phosphorus (designated as PO\u003csub\u003e3\u003c/sub\u003e-DH-DG in Fig. 2B). Upon GC-MS analysis of carbohydrate in the form of 1-O-methyl 2,3,4,6-tetra-O-trimethylsilyl derivatives after methanolysis, glucose was found to be the only carbohydrate in all glycolipids from PP and TH. As shown in Fig. 3 and Table 2, partially methylated aldohexitol acetates prepared from DH-DG in PP gave two peaks, whose retention times and mass spectra were identical to those of 1,5-di-O-acetyl 2,3,4,6-tetra-O-methyl Glc (terminal Glc) for peak a and 1,2,5-tri-O-acetyl 3,4,6-tri-O-methyl Glc (2-linked Glc) for peak b. In addition, \u003csup\u003e1\u003c/sup\u003eH-NMR spectra (Fig. 3C) contained doublet signals at\u0026nbsp;d=4.913 ppm (J=3.2 Hz) and\u0026nbsp;d=4.821 ppm (J=4.0 Hz), indicating that the anomers of both of the two Glc residues in DH-DG in PP were in the\u0026nbsp;a-configuration. Accordingly, the structure of DH-DG from PP was shown to be Glca1-2Glca1-3¢DG, namely, kojibiosyl DG. Identical results were obtained for DH-DG from TH, whose structure was also determined to be Glca1-2Glca1-3¢DG (Table 2). Thus, the carbohydrate structures of DH-DG in \u003cem\u003ePediococcus\u003c/em\u003e and \u003cem\u003eTetragenococcus\u003c/em\u003e species were shown to be the same as those in \u003cem\u003eStreptococcus\u003c/em\u003e species (StrDH-DG). The positive-ion FABMS spectra gave the abundant molecular ions [DH-DG+Na]\u003csup\u003e+\u003c/sup\u003e, m/z 967 (74.9%) for 18:1 and 18:1 from DH-DG of PP, and m/z 941 (44.6%) for 16:0 and 18:1 from DH-DG of TH (Fig. 4, Table 3). The glucose residues of MH-DG in both bacteria (Table 2) were susceptible to\u0026nbsp;a-glucosidase (rice) to yield DG, although the\u0026nbsp;a-1,2-linkage of the terminal Glc in DH-DG was not cleaved by\u0026nbsp;a-glucosidase, which reacted with\u0026nbsp;a1,4- and\u0026nbsp;a1,6-linkages of Glc in oligosaccharides. Thus, MH-DGs in both PP and TH were suggested to be Glca1-3¢DG, whose molecular species were similar to those of DH-DG (Table 3). Meanwhile, 2-linked and 6-linked Glcs were detected from both PO\u003csub\u003e3\u003c/sub\u003e-DH-DG of PP and TH at a ratio of 1:1, indicating a modification of the 6-position of the terminal Glc of DH-DG (Table 2). The negative-ion FABMS spectra gave molecular ions of [M-H]\u003csup\u003e-\u003c/sup\u003e, m/z 1097, from PO\u003csub\u003e3\u003c/sub\u003e-DH-DG (18:1,18:1) of PP and m/z 1071 from PO\u003csub\u003e3\u003c/sub\u003e-DH-DG (16:0,18:1) of TH, suggesting that glycerol phosphate (Gro-P, molecular weight of 155) is attached to the 6-position of terminal Glc (Fig. 4, Table 3). The structures of PO\u003csub\u003e3\u003c/sub\u003e-DH-DG of both PP and TH were suggested to be 6-(Gro-PO\u003csub\u003e3\u003c/sub\u003e)-Glca1-2Glca1-3¢DG, which was already reported as a typical modified structure of kojibiosyl DG in Gram-positive bacteria [8,9].\u003c/p\u003e\n\u003cp\u003eAmounts of phospholipids and glycolipids in PP and TH\u003c/p\u003e\n\u003cp\u003eOn the basis of the data obtained by TLC densitometry, the amounts of CL, PG, and glycolipids in PP and TH were estimated, as shown in Table 4. The major phospholipids, CL and PG, amounted to 2.0\u0026nbsp;mg/mg dry weight for PP and 2.75\u0026nbsp;mg/mg dry weight for TH and the ratios of CL to PG were approximately 1:2 for both bacteria. The total amounts of glycolipids were 1.45\u0026nbsp;mg/mg dry weight for PP and 1.9\u0026nbsp;mg/mg dry weight for TH, of which Glca1-2Glca1-3¢DG (DH-DG) comprised 65.5% and 81.6% of the total, respectively. Thus, the lipid bilayers of both PP and TH consisted of CL, PG, and DH-DG.\u003c/p\u003e\n\u003cp\u003eAntibodies for bacterial DH-DG in human sera\u003c/p\u003e\n\u003cp\u003eHuman sera of blood groups A, B, AB, and O were reacted with LacDH-DG, StaDH-DG, StrDH-DG, and Forssman glycolipids on an ELISA plate, and those giving optical densities in excess of 0.4 were able to generate positive bands for the respective glycolipids by TLC immunostaining (Table 5). The antibodies toward Forssman antigens were frequently detected in human sera (in 16 out of 20 sera), irrespective of the ABO blood group. As shown in Fig. 5A, high-performance TLC (HPTLC) revealed that the mobility of DH-DG from PP was identical to that of StrDH-DG from SS, but that from TH was slightly lower than that of StrDH-DG from SS, owing to the higher amount of palmitic acid in its DG moiety from TH. In addition, StaDH-DG with ai15:0 and ai17:0 from SE migrated to a position lower than DH-DG from TH. Meanwhile, all DH-DGs on the plastic TLC plate had the same mobility \u0026nbsp; \u0026nbsp;(Fig. 5B). Serum samples 3 and 13, and Serum samples 5 and 13, were found to contain antibodies for LacDH-DG and StaDH-DG, respectively (Fig. 5B). In particular, since serum 13 was from a patient who had suffered fromfood poisoning due to \u003cem\u003eStaphylococcus aureus\u003c/em\u003e 6 months previously, antibodies for StaDH-DG (gentibiosyl DG) were thought to have been generated by staphylococcal infection. Moreover, the optical densities of antibody binding to StrDH-DG of all sera examined by ELISA were lower than 0.17, and the binding of antibodies to StrDH-DG was not detected by TLC immunostaining.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUpon comparing the glycolipid structures of symbiotic bacteria living in the human oral cavity, intestine, and skin, they were each shown to have characteristic fatty acid compositions of their DG moieties, as potential adaptations to the completely different environmental conditions that prevail at these anatomical locations. Meanwhile, those from PP and TH were composed of unsaturated fatty acids as more than 70% of the total molecular species, resembling the findings for SS in the oral cavity. Moreover, the carbohydrate structures of DH-DGs were characteristic of the particular bacterial species, providing a landmark for the bacterial species to distinguish each other and probably for their hosts to recognize them. The disaccharide structures of DH-DGs in \u003cem\u003ePediococcus\u003c/em\u003e and \u003cem\u003eTetragenococcus\u003c/em\u003e species were kojibiose, in which two α-glucoses are linked by an α1\u0026ndash;2 linkage, and were also identical to StrDH-DG in SS in the oral cavity. Since α-glucose is a constituent of glycogen and starch as the major nutrients in humans, the intestinal immune system is continuously exposed to it to ensure tolerance to it. This could help to explain why SS can inhabit the oral cavity as a symbiotic bacterium, and why PP and TH were selected as bacteria for the preparation of fermented foods, namely, miso and soy sauce, respectively. In addition, although α1\u0026ndash;4 and α1\u0026ndash;6 linkages of Glc in glycogen and starch are readily hydrolyzed by α-glucosidase, the α1\u0026ndash;2 linkage is resistant to enzymatic cleavage, so kojibiose is used as an inhibitor of α-glucosidase [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, the glycosidic linkage in StrDH-DG in SS, PP, and TH might not be hydrolyzed during the digestion process, despite bacterial mortality due to the strongly acidic conditions in the stomach, although monoglucosyl DG was reportedly hydrolyzed to yield Glc and DG by α-glucosidase [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In contrast, StrDH-DG, as a derivative of kojibiose linked with DG, did not inhibit the activity of α-glucosidase (rice) with \u003cem\u003ep\u003c/em\u003e-nitrophenyl α-D-glucopyranoside as the substrate. This showed that the enzymatic breakdown of starch and glycogen by α-glucosidase is not affected by StrDH-DG in SS, PP, and TH (Iwamori et al., unpublished observation). Similarly, the terminal carbohydrate linkages of DH-DGs in LJ and SE (i.e., Galα1-2 of LacDH-DG and Glcβ1-6 of StaDH-DG) were thought to be resistant to digestion because of the absence of exoglycosidases for them in the digestive tract. In fact, LacTetH-DG, LacTH-DG, and LacDH-DG in LJ were reportedly maintained without degradation in the cecal and colonic contents and feces of mice [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the case of LJ, Gg\u003csub\u003e4\u003c/sub\u003eCer in the duodenal, jejunal, and ileal epithelia of the murine digestive tract was reported as a receptor enabling the formation of intestinal bacterial flora [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and was also excreted into the feces without degradation. This indicates that Gg\u003csub\u003e4\u003c/sub\u003eCer in the feces might facilitate the discharge of LJ and other bacteria using it as a receptor, by attaching to them to prevent their irregular diffusion upon their release from intestinal epithelia into the digestive tract [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In fact, upon TLC immunostaining with anti-LJ, anti-SE, and anti-SS antisera of lipid extracts from the feces of 2-month-old female mice, although several bands appeared in the regions of MH-DG and DH-DG and could not be distinguished among them, LacTH-DG and LacTetH-DG were only detected with anti-LJ antisera, at amounts corresponding to approximately 1.4 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells/g dry weight [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, Gg\u003csub\u003e4\u003c/sub\u003eCer, corresponding to approximately 20% of the total amounts of Gg\u003csub\u003e4\u003c/sub\u003eCer in the duodenal, jejunal, and ileal epithelia, was excreted into the feces without degradation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGlycolipids are generally known to be strongly antigenic compounds, and antibodies against their carbohydrate moieties are readily generated by epidermal immunization with tissues and cells, as well as with purified glycolipids [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, bacterial glycolipids in the digestive tract are expected to be recognized as foreign antigens to yield circulating antibodies in sera through their processing by gut-associated lymphoid tissues in Peyer\u0026rsquo;s patches [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Among glycolipids in LJ, SE, and SS, those with three or four carbohydrates, namely, LacTH-DG and LacTetH-DG, generated antibodies at greater intensity and higher specificity than those with one or two carbohydrates, upon direct immunization of rabbits with bacteria together with Freund\u0026rsquo;s complete adjuvant [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In practice, antibodies against LacTH-DG and LacTetH-DG in LJ were detected in human sera at frequencies and intensities similar to those against ABO-blood group-related glycolipids [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Since LacTH-DG and LacTetH-DG are contained in various \u003cem\u003eLactobacillus\u003c/em\u003e species and strains, continuous stimulation of the gut-associated lymphoid tissues by intestinal symbiotic bacteria and bacteria in fermented foods might result in the production of antibodies for them. However, it has remained unknown whether such antibodies play a role in preventing bacteria from invading the bloodstream. Interestingly, anti-glycolipid antibodies generated upon infection with \u003cem\u003eCampylobacter jejuni\u003c/em\u003e are known to be closely associated with the onset of autoimmune diseases, such as Guillain\u0026ndash;Barr\u0026eacute; and Miller\u0026ndash;Fisher syndromes [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the case of \u003cem\u003eC. jejuni\u003c/em\u003e as a Gram-negative bacterium, the terminal structures of lipooligosaccharides were identical to those of glycosphingolipids in human neural tissues [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], resulting in the production of antibodies that cross-react with glycosphingolipids, followed by antibody-mediated inflammatory neuropathies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, since structures mimicking Forssman and ABO-blood group-related oligosaccharides have yet to be detected in any bacteria, the original stimuli generating the antibodies against them are obscure, although several hypotheses explaining the production of natural antibodies have been proposed [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, antibodies against DH-DG, with a rather short carbohydrate chain, were thought to be generated by abnormal stimulation of the respective bacteria. As shown in this paper, anti-StaDH-DG antibodies were detected in 2 out of 20 human sera, one of which was from a patient who had suffered from food poisoning due to \u003cem\u003eS. aureus\u003c/em\u003e 6 months previously. It is likely that, although \u003cem\u003eS. epidermidis\u003c/em\u003e as a symbiotic bacterium normally stimulates the immune system in the skin on a daily basis, abnormal stimulation by \u003cem\u003eS. aureus\u003c/em\u003e might cause the production of antibodies against StaDH-DG (gentibiosyl DG). To test this hypothesis, there is a need for further examinations with a large number of human serum samples in order to clarify the correlation between anti-DH-DG antibodies and disorders of the intestinal microbiota.\u003c/p\u003e \u003cp\u003eIn addition, α-galactosyl ceramide from marine sponge and α-galacturonyl ceramide from \u003cem\u003eSphingomonas\u003c/em\u003e bacteria were revealed to exert immunoregulatory activity on CD1d-restricted natural killer T cells [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. It is likely that bacterial glycolipids possessing similar bioactivity are found in the Gram-positive bacteria inhabiting various anatomical sites of the human body. A systematic analysis of glycolipid structures in various bacterial species is required to understand glycolipid signaling between bacteria and immunocytes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eThe nomenclature for glycosphingolipids is based on the recommendations of the IUPAC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIUB Commission on Biochemical Nomenclature [1]\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDG, diglyceride; Gro, glycerol; LacDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDG, \u003cem\u003eLactobacillus\u003c/em\u003e dihexaosyl diglyceride, Galα1-2Glcα1-3\u0026prime;DG; StrDH-DG, \u003cem\u003eStreptococcus\u003c/em\u003e dihexaosyl diglyceride, Glcα1-2Glcα1-3\u0026prime;DG; StaDH-DG, \u003cem\u003eStaphylococcus\u003c/em\u003e dihexaosyl diglyceride, Glcβ1-6Glcβ1-3\u0026prime;DG; PG, phosphatidyl glycerol; CL, cardiolipin; TLC, thin layer chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompliance with ethical standards\u003c/h2\u003e \u003cp\u003eConflict of interest\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthical approval\u003c/h2\u003e \u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003esupport: no\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBoth authors equally contributed for this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIUPAC-IUB Commission on Biochemical Nomenclature: The Nomenclature of Lipids. Eur. J. Biochem. \u003cstrong\u003e179\u003c/strong\u003e, 11-21 (1977)\u003c/li\u003e\n\u003cli\u003eIwamori, M., Nakasa, M., Yamazaki, K., Iwamori, Y., Tanaka, K., Aoki, D., Adachi, S., Nomura, T.: Bacterial species-characteristic profiles of molecular species, and the antigenicity of phospholipids and glycolipids in symbiotic Lactobacillus, Staphylococcus and Streptococcus species. Glycoconj. J. \u003cstrong\u003e29\u003c/strong\u003e, 199-209 (2012)\u003c/li\u003e\n\u003cli\u003eShaw, N.: Bacterial glycolipids. Bacteriol. Rev. \u003cstrong\u003e34\u003c/strong\u003e, 365-77 (1970)\u003c/li\u003e\n\u003cli\u003eIwamori, M., Sakai, A., Minamimoto, N., Iwamori, Y., Tanaka, K., Aoki, D., Adachi, S., Nomura, T.: Characterization of novel glycolipid antigens with an \u0026alpha;-galactose epitope in lactobacilli detected with rabbit anti-Lactobacillus antiserum and occurrence of antibodies against them in human sera. J. Biochem. \u003cstrong\u003e150\u003c/strong\u003e, 515-523 (2011) \u003c/li\u003e\n\u003cli\u003eIwamori, M., Adachi, S., Lin, B., Tanaka, K., Aoki, D. and Nomura, T.: Spermatogenesis-associated changes of fucosylated glycolipids in murine testis. Human Cell \u003cstrong\u003e33\u003c/strong\u003e, 23-28 (2020)\u003c/li\u003e\n\u003cli\u003eIwamori, M., Tanaka, K., Adachi, S., Aoki, D., Nomura, T.: Absence of lactobacilli containing glycolipids with the \u0026alpha;-galactose epitope and the enhanced fucosylation of a receptor glycolipid GA1 in the digestive tracts of immune-deficient scid mice. J. Biochem. \u003cstrong\u003e158\u003c/strong\u003e, 73-82 (2015) \u003c/li\u003e\n\u003cli\u003eOhata, E,, Yoshida, S., Masuda, T., Kitakawa, M., Nakazawa, T., Okada, M., Yasui, H.: \u003cem\u003eTetragenococcus halophilus\u003c/em\u003e MN45 isolated from miso inhibits IgE production. Food Sci. Tech. \u003cstrong\u003e17\u003c/strong\u003e, 129-138 (2011)\u003c/li\u003e\n\u003cli\u003eFischer, W., Laine, R.A., Nakano, M.: On the relationship between glycerophosphoglycolipids and lipoteichoic acid in Gram-positive bacteria. II Structures of glycerophosphoglycolipids. Biochim. Biophys. Acta \u003cstrong\u003e528\u003c/strong\u003e, 298-308 (1978)\u003c/li\u003e\n\u003cli\u003eFischer, W., Schuster, D., Laine, R.A.: Studies on the relationship between glycerophosphoglycolipids and lipoteichoic acids. IV Trigalactosyl glycerophospho-acylkojibiosyldiacylglycerol and related compounds from \u003cem\u003eStreptococcus lactis Kiel\u003c/em\u003e 42172. Biochim. Biophys. Acta \u003cstrong\u003e575\u003c/strong\u003e, 389-398 (1979)\u003c/li\u003e\n\u003cli\u003eLaparra, J.M., Diez-Municio, M., Moreno, F.J., Herreno, M.: Kojibiose ameliorates arachidic acid-induced metabolic alterations in hyperglycaemic rats. Br. J. Nutr. \u003cstrong\u003e114\u003c/strong\u003e, 1395-1402 (2015)\u003c/li\u003e\n\u003cli\u003eForsgard, R.A., Lactose digestion in humans: intestinal lactose appears to be constitutive whereas the colonic microbiome is adaptable. Am. J. Clin. Nutr. \u003cstrong\u003e110\u003c/strong\u003e, 273-279 (2019)\u003c/li\u003e\n\u003cli\u003eIwamori, M., Iwamori, Y., Adachi, S., Nomura, T. Excretion into faeces of asialoGM1 in the murine defective tract and \u003cem\u003eLactobacillus johnsoni\u003c/em\u003e exhibiting binding ability toward asialoGM1. A possible role of epithelial glycolipids in the discharge of intestinal bacteria. Glycoconj. J. \u003cstrong\u003e28\u003c/strong\u003e, 21-30 (2011)\u003c/li\u003e\n\u003cli\u003eKarlsson, K.A. Animal glycoshingolipids as membrane attachment sites for bacteria. Ann. Rev. Biochem. \u003cstrong\u003e58\u003c/strong\u003e, 309-350 (1989)\u003c/li\u003e\n\u003cli\u003eIwamori, M. A new turning point in glycosphingolipid research. 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R.: The development and consequences of red blood cell alloimmunization. Annu. Rev. Pathol. \u003cstrong\u003e18\u003c/strong\u003e, 537-564 (2023)\u003c/li\u003e\n\u003cli\u003eAvci, F.Y., Kasper, D.L.: How bacterial carbohydrate influence the adaptive immune system. Annu. Rev. Immunol. \u003cstrong\u003e28\u003c/strong\u003e, 107-130 (2010)\u003c/li\u003e\n\u003cli\u003eNishimura, T., Kitamura, H., Iwakabe, K., Yahata, T., Ohta, A., Sato, M., Takeda, K., Okumura, K., Vankaer, L., Kawano, T., Taniguchi, M., Nakui, M., Sekimoto, M., Koga, T.: The interface between innate and acquired immunity: glycolipid antigen presentation by CD1d-expressing dendritic cells to NKT cells induce the differentiation of antigen-specific cytotoxic T lymphocytes. Int. Immunol. \u003cstrong\u003e12\u003c/strong\u003e, 987-994 (2000)\u003c/li\u003e\n\u003cli\u003eWu, D., Xing, G., Poles, M.A., Wong, C.: Bacterial glycolipids and analogs as antigens for CD1d-restricted NKT cells. Proc. Natl. Acad. Sci. USA \u003cstrong\u003e102\u003c/strong\u003e, 1351-1356 (2005)\u003c/li\u003e\n\u003cli\u003eAbdel-Mawgoud, A. M.: Stephanopoulos,G., Simple glycolipids of microbes: Chemistry, biological activity and metabolic engineering. Synth. Syst. Biotechnol. \u003cstrong\u003e15\u003c/strong\u003e, 3-19 (2017)\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1-5 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"glycoconjugate-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"glyc","sideBox":"Learn more about [Glycoconjugate Journal](http://link.springer.com/journal/10719)","snPcode":"10719","submissionUrl":"https://submission.nature.com/new-submission/10719/3","title":"Glycoconjugate Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"bacterial dihexaosyl diglyceride, kojibiose, bacteria for fermented food, symbiotic bacteria, anti-glycolipid antibody, TLC immunostaining","lastPublishedDoi":"10.21203/rs.3.rs-6265412/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6265412/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGram-positive bacterial biomembranes are composed of phosphatidyl glycerol (PG), cardiolipin (CL), and dihexaosyl diglycerides (DH-DG) as the major lipid constituents. The carbohydrate structures of DH-DG are specific to the particular bacterial species and we previously revealed them to have immunologically active properties. To characterize the functional significance of glycolipids in Gram-positive bacteria for fermented foods, the structures of DH-DG in \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e (PP) for producing fermented soybean paste (miso) and \u003cem\u003eTetragenococcus halophilus\u003c/em\u003e (TH) for soy sauce were determined. They were shown to be Glcα1-2Glcα1-3\u0026prime;DG (kojibiosyl DG) with 18:1 and 18:1 for PP, and those with 16:0 and 18:1 for TH, both of which were identical to DH-DG from \u003cem\u003eStreptococcus salivarius\u003c/em\u003e, a symbiotic bacterium in the human oral cavity. Additionally, both bacteria contained an acidic glycolipid, in which glycerol phosphate was attached to the 6-position of the nonreducing terminal Glc residue of DH-DG. DH-DG in PP and TH comprised 65.5% and 81.6% of the total glycolipids, respectively, and the ratios of PG and DH-DG to CL were 1.9 and 1.4 for PP, and 2.0 and 1.7 for TH. TLC immunostaining with human sera revealed antibodies for Galα1-2Glcα1-3\u0026prime;DG (LacDH-DG) from \u003cem\u003eLactobacillus\u003c/em\u003e species and Glcβ1-6Glcβ1-3\u0026prime;DG (StaDH-DG) from \u003cem\u003eStaphylococcus\u003c/em\u003e species, but not for Glcα1-2Glcα1-3\u0026prime;DG (StrDH-DG) from \u003cem\u003eStreptococcus\u003c/em\u003e species, in 2 out of 20 human sera. Given that one serum sample with anti-StaDH-DG antibodies was from a patient who had suffered food poisoning due to \u003cem\u003eStaphylococcus aureus\u003c/em\u003e 6 months previously, the antibodies for bacterial DH-DG were thought to have arisen via bacterial infection.\u003c/p\u003e","manuscriptTitle":"Bacterial glycolipids in Pediococcus pentosaceus for fermented soybean paste (miso) and Tetragenococcus halophilus for soy sauce","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 09:00:03","doi":"10.21203/rs.3.rs-6265412/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-13T15:09:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-12T02:19:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59688105835315242162564901250413041787","date":"2025-06-10T02:27:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-06T02:33:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22675150773747329037987073247982076436","date":"2025-03-27T23:22:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-27T13:26:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-27T09:52:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-27T09:52:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Glycoconjugate Journal","date":"2025-03-20T02:16:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"glycoconjugate-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"glyc","sideBox":"Learn more about [Glycoconjugate Journal](http://link.springer.com/journal/10719)","snPcode":"10719","submissionUrl":"https://submission.nature.com/new-submission/10719/3","title":"Glycoconjugate Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7613c82a-bc7b-4e27-a29f-7238e3ad1880","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-21T16:08:57+00:00","versionOfRecord":{"articleIdentity":"rs-6265412","link":"https://doi.org/10.1007/s10719-025-10190-1","journal":{"identity":"glycoconjugate-journal","isVorOnly":false,"title":"Glycoconjugate Journal"},"publishedOn":"2025-07-15 16:05:40","publishedOnDateReadable":"July 15th, 2025"},"versionCreatedAt":"2025-04-16 09:00:03","video":"","vorDoi":"10.1007/s10719-025-10190-1","vorDoiUrl":"https://doi.org/10.1007/s10719-025-10190-1","workflowStages":[]},"version":"v1","identity":"rs-6265412","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6265412","identity":"rs-6265412","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}