Characterization of novel glycolipid antigens with an  -galactose epitope in lactobacilli detected with rabbit anti-Lactobacillus antisera and occurrence of antibodies against them in human sera

Abstract

Anti-Lactobacillus johnsonii (LJ) antisera generated by immunization of rabbits with LJ reacted with glyceroglycolipids in LJ, i.e. dihexaosyl diacylglycerol (DH-DG), trihexaosyl DG (TH-DG) and tetrahexaosyl DG (TetH-DG), whose reactivities with antisera increased proportionally with longer carbohydrate chains of glycolipids. Structural analyses of glycolipids from LJ revealed that DH-DG was Galα1-2Glcα1-3'DG, and TH-DG and TetH-DG were novel derivatives of it with α-Gal at the non-reducing terminal, i.e. Galα1-6Galα1-2Glcα1-3'DG and Galα1-6Galα1-6Galα1-2Glcα1-3'DG, respectively. DH-DG was commonly present in several lactobacilli examined, but TetH-DG was restricted to LJ, L. intestinalis and L. reuteri, while the TH-DGs from L. casei were Glc1-6Galα1-2Glcα1-3'DG and an esterified derivative of it, Glc1-6Galα1-2Glc(6-fatty acid)α1-3'DG, as reported in the literature. Anti-LJ antisera reacted with TH-DG and esterified TH-DG from L. casei to lesser extents, but not at all with gentibiosyl DG from Staphylococcus epidermidis or kojibiosyl DG from Streptococcus salivalis or sphingoglycolipids containing α-Gal residues. The major molecular species of glycolipids obtained from lactobacilli were 11-octadecenoic and 11,12-methylene-octadecanoic acids-containing ones. Also, human IgM antibodies against TH-DG and TetH-DG from LJ were detected in human sera, with various antibody titres, indicating that an immune reaction to symbiotic lactobacilli occurs against their glycolipid antigens, TH-DG and TetH-DG.

Bacterial species-characteristic profiles of molecular species, and the antigenicity of phospholipids and glycolipids in symbiotic Lactobacillus, Staphylococcus and Streptococcus species

“…As reported previously [2], the major lipid constituents of Gram-positive bacteria belonging to the Lactobacillus species are glycerophospholipids and glyceroglycolipids, among which dihexaosyl diglycerides (DH-DG) are present in concentrations equivalent to those of phospholipids. However, the carbohydrate structure of DH-DG is known to be bacterial species-specific, and its modification occurs in individual strains of the same bacterial species.…”

“…at 60°C for 5 min, followed by analysis with a GC-MS (GP5050: Shimadzu, Kyoto) equipped with a DB-1 column (0.25 mmø x 30 m) from 150°C to 250°C at 10°C/min. Also, linkage analysis of carbohydrates was carried out by conversion of glycolipids to partially methylated aldohexitol acetates, followed by analysis with a GC-MS with a DB-1 column from 150°C to 210°C at 4°C/min [2][3][4]. In addition, for determination of the carbohydrate sequences and anomeric configurations, glycolipids were hydrolyzed with the following glycosidases: α-glucosidase (rice; Sigma) in 50 mM citrate buffer (pH 4.5), β-glucosidase (almond; Wako, Tokyo) in 50 mM citrate buffer (pH 6.5), and α-galactosidase (coffee bean; Sigma) in 50 mM citrate buffer (pH 4.5).…”

“…All Lactobacillus strains examined contained Galα1-2Glcα1-3′DG as a Lactobacillus species-characteristic glycolipid, and triand tetrahexaosyl DGs, in which α-Gal is linked at the nonreducing terminal of Lactobacilli-characteristic DH-DG through an α1-6 linkage, have been detected in the intestinal Lactobacilli of several animal species, i.e. L.johnsonii (LJ) and L. intestinalis, but not in non-intestinal L. fermentum and L. plantarum [2]. On immunization of rabbits with LJ, antibodies against Galα1-2Glcα1-3′DG, Galα1-6Galα1-2Glcα1-3′DG (TH-DG), and Galα1-6Galα1-6Galα1-2Glcα1-3′DG (TetH-DG), but not against phospholipids, have been readily yielded, indicating their strong antigenicities.…”

“…Since anti-LJ antisera exhibit sufficiently high specificity and titers toward these glycolipids, particularly toward TH-DG and TetH-DG, they have been used for the sensitive detection and quantitation of bacteria belonging to the Lactobacillus species in intestinal contents [3,4]. In addition, antibodies to them have been detected in normal human sera, indicating that human immune surveillance ability as to intestinal symbiotic Lactobacilli is partly gained through the recognition of bacterial glycolipids as target antigens [2]. To further characterize the antigenic properties of bacterial glycolipids in relation with symbiosis, antisera against Staphylococcus epidermidis (SE) in skin and Streptococcus salivalis (SS) in the oral cavity were generated by direct immunization of rabbits with them.…”

“…cyclopropane ring-containing cy19:0 fatty acid (11,12-methylene-octadecanoic acid) and anteiso-type (ai15:0 and ai17:0) fatty acids, respectively [2]. In general, the hydrophobic chains of lipids in mammalian tissues and cells are known to differ between glycolipids and phospholipids, and between phospholipids distributed in the inner and outer leaflets of the lipid bilayer [5][6][7].…”