Abstract
BackgroundBifidobacterium longum 105-A produces markedly high amounts of capsular polysaccharides (CPS) and exopolysaccharides (EPS) that should play distinct roles in bacterial–host interactions. To identify the biological function of B. longum 105-A CPS/EPS, we carried out an informatics survey of the genome and identified the EPS-encoding genetic locus of B. longum 105-A that is responsible for the production of CPS/EPS. The role of CPS/EPS in the adaptation to gut tract environment and bacteria-gut cell interactions was investigated using the ΔcpsD mutant.ResultsA putative B. longum 105-A CPS/EPS gene cluster was shown to consist of 24 putative genes encoding a priming glycosyltransferase (cpsD), 7 glycosyltransferases, 4 CPS/EPS synthesis machinery proteins, and 3 dTDP-L-rhamnose synthesis enzymes. These enzymes should form a complex system that is involved in the biogenesis of CPS and/or EPS. To confirm this, we constructed a knockout mutant (ΔcpsD) by a double cross-over homologous recombination. Compared to wild-type, the ∆cpsD mutant showed a similar growth rate. However, it showed quicker sedimentation and formation of cell clusters in liquid culture. EPS was secreted by the ∆cpsD mutant, but had altered monosaccharide composition and molecular weight. Comparison of the morphology of B. longum 105-A wild-type and ∆cpsD by negative staining in light and electron microscopy revealed that the formation of fimbriae is drastically enhanced in the ∆cpsD mutant while the B. longum 105-A wild-type was coated by a thick capsule. The fimbriae expression in the ∆cpsD was closely associated with the disappearance of the CPS layer. The wild-type showed low pH tolerance, adaptation, and bile salt tolerance, but the ∆cpsD mutant had lost this survivability in gastric and duodenal environments. The ∆cpsD mutant was extensively able to bind to the human colon carcinoma Caco-2 cell line and was phagocytosed by murine macrophage RAW 264.7, whereas the wild-type did not bind to epithelial cells and totally resisted internalization by macrophages.ConclusionsOur results suggest that CPS/EPS production and fimbriae formation are negatively correlated and play key roles in the survival, attachment, and colonization of B. longum 105-A in the gut.
Highlights
Bifidobacterium longum 105-A produces markedly high amounts of capsular polysaccharides (CPS) and exopolysaccharides (EPS) that should play distinct roles in bacterial–host interactions
Identifying of EPS locus and annotation of the putative EPS biosynthetic cluster in B. longum 105‐A The genome of B. longum 105-A [61] harbors a putative EPS-encoding locus, which extends from BL105A-0403 to BL105A-0427 and encompasses a 33.2-kb region that harbors 24 genes predicted to be involved in EPS biosynthesis (Fig. 1; Table 1), four genes (BL105A_0403, BL105A_0419, BL105A_0421, and BL105A_0422) that encode a putative transposase, and three genes (BL105A_0418, BL105A_0421, and BL105A_0423) that encode a putative integrase
The EPS gene cluster of B. longum 105-A is modular in the organization, which is commonly reported for surface heteropolysaccharides [63]
Summary
Identifying of EPS locus and annotation of the putative EPS biosynthetic cluster in B. longum 105‐A The genome of B. longum 105-A [61] harbors a putative EPS-encoding locus, which extends from BL105A-0403 to BL105A-0427 and encompasses a 33.2-kb region that harbors 24 genes predicted to be involved in EPS biosynthesis (Fig. 1; Table 1), four genes (BL105A_0403, BL105A_0419, BL105A_0421, and BL105A_0422) that encode a putative transposase, and three genes (BL105A_0418, BL105A_0421, and BL105A_0423) that encode a putative integrase. We observed that in the identified putative B. longum 105-A EPS gene cluster, the genes which encode the putative priming-glycosyltransferase, envelope protein, chain length determinant protein, first two glycosyltransferases, rhamnose biosynthesis precursors, and tyrosine kinase (BL105A_0405–BL105A_0409, BL105A_0424– BL105A_0427) are conserved in other Bifidobacterium strains, but other glycosyltransferases, polymerase, pyruvyltransferase, flippase, and acetyltransferase genes (BL105A_0410–BL105A_0417) are specific for the associated EPS repeating unit (36–37), as reflected by their lower level of similarity to other orthologs (Table 1), according to the reported mechanism of EPS production in S. pneumoniae and L. rhamnosus [64, 69]. This study demonstrates that the wildtype B. longum 105-A did not bind to the epithelial cells in comparison to extensive binding by its cpsD mutant (Fig. 5a, b) These results were completely different from that demonstrated by Fanning et al 2012, which showed a significant role of CPS in the initial binding of B. breve in the initial colonization of the mouse gut. Further investigations, including in vivo study, should be carried out to examine its ability to survive in the animal host and to its role in immune modulation and preventing the infection with pathogenic microorganisms
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