Enzymatically synthesized exopolysaccharide of a probiotic strain Leuconostoc mesenteroides NTM048 shows adjuvant activity to promote IgA antibody responses

Abstract

Leuconostoc mesenteroides strain NTM048 produces an exopolysaccharide (EPS; glucose polymers 94% and fructose polymers 6%) with adjuvanticity for mucosal vaccination. Strain NTM048 includes three putative EPS-synthesizing genes, gtf1 and gtf2 for synthesizing glucose polymers, and lvnS for synthesizing fructose polymer. To elucidate the key polymer structure for adjuvanticity, two genes, gtf1 and gtf2, which were annotated as glycoside hydrolase family 70 enzyme genes, were expressed in Escherichia coli. Glycosyl-linkage composition analysis and NMR analysis showed that the recombinant enzyme Gtf1 produced a soluble form of α-1,6-glucan, whereas the recombinant enzyme Gtf2 produced glucans with approximately equal percentages of α-1,6-and α-1,3-glucose residues both in the supernatant (S-glucan) and as a precipitate (P-glucan). Comparison of polysaccharides synthesized by Gtf1, Gtf2, and LvnS revealed that Gtf2-S-glucan, which was produced in the supernatant by Gtf2 and formed particles of 7.8 µm, possessed 1.8-fold higher ability to stimulate IgA production from murine Peyer's patch cells than native NTM048 EPS. Evaluation of adjuvanticity by intranasal administration of mice with an antigen (ovalbumin) and Gtf2-S-glucan or NTM048 EPS showed that Gtf2-S-glucan induced the production of higher antigenspecific antibodies in the airway mucosa and plasma, suggesting a pivotal role of Gtf2-S-glucan in the adjuvanticity of NTM048 EPS.

Chemical Synthesis and Evaluation of Exopolysaccharide Fragments Produced by <i>Leuconostoc mesenteroides</i> Strain NTM048

“…6) NMR and GC-MS analyses of methylated derivatives of EPSs revealed that NTM048 EPS is a dextran consisting of an α-(1→ 6)-linked glucan with 4% α-(1→ 3) branches, and the remaining fructose polymer is composed of β-fructans. 7) Our group also found that enzymatically synthesized β-fructans exhibited lower IgA-inducing activity than NTM048 EPS, whereas α-glucans synthesized by a GH70 enzyme cloned from strain NTM048 possessed stronger IgA-inducing activity than NTM048 EPS. 8) These results indicate that α-glucans play an important role in the induction of IgA.…”

“…The characteristic peak at the branching point of the Glc(3,6-Glc) of fragment 3 (H-1 of branched-Glc: 5.32 ppm, J 1,2 = 4.0 Hz) was consistent with that of NTM048 EPS (H-1 of branched-Glc: 5.32 ppm, J 1,2 = 3.9 Hz). 7) With fragments 2 and 3 in hand, we next evaluated their IgA-inducing ability using murine Payer's patch cells. 8) In this assay, α-glucans synthesized by Gtf1 and Gtf2 were also used as controls in addition to NTM048 EPS.…”

Relationship of Gene-Structure–Antioxidant Ability of Exopolysaccharides Derived from Lactic Acid Bacteria: A Review

“…20 ■ HOPS SYNTHESIS BY LAB HoPS consist of one monosaccharide�glucose or fructose� and are generally produced by the generas Weissella, Leuconostoc, Lactobacillus, and Pediococcus via the extracellular synthetic pathway. The process is mediated by specific glycosyltransferases (GTFs) and fructotransferases (FTFs) 21,22 and consists of two steps. In the first step, the sugar residues are cleaved into monomeric units and assembled into polymers with the aid of GTFs.…”

Strategies for applying probiotics in the antibiotic management of Clostridioides difficile infection

“…69 Whether it is a traditional probiotic or a new live biologic, mainstream clinical use has chosen to use probiotics after antibiotics, and there may be multiple reasons for such a combination order (Fig. 1): (1) the subsequent addition of probiotics can effectively occupy these ecological sites, thus competing to exclude the more resistant C. difficile strains, vegetative cells and spores; 9 (2) the addition of probiotics competes with the remaining C. difficile strains and their vegetative cells for nutrients essential for growth such as carbohydrates and amino acids, thus inhibiting the normal growth and metabolism of C. difficile; 19 (3) probiotics are able to secrete organic acids after colonization and affect the level of intestinal bile acid metabolism, especially enhancing the level of secondary bile acids, thus altering the intestinal growth environment to the detriment of C. difficile; 49,50 (4) after colonization, probiotics can secrete antimicrobial peptides, bacteriocins, extracellular polysaccharides and other types of antimicrobial substances, which have a direct killing effect on C. difficile strains; 64,[70][71][72] (5) probiotics can activate the intestinal immune system and enhance the related immune response to clear C. difficile; 73 and (6) probiotics can indirectly promote the growth of low-abundance beneficial microbes in the gut by "cross-feeding", thus co-antagonizing C. difficile. 74 Fig.…”