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. 2017 May 1:9:27.
doi: 10.1186/s13099-017-0177-x. eCollection 2017.

Capsular polysaccharide inhibits adhesion of Bifidobacterium longum 105-A to enterocyte-like Caco-2 cells and phagocytosis by macrophages

Amin Tahoun #  1   2 , Hisayoshi Masutani #  1 , Hanem El-Sharkawy  2   3 , Trudi Gillespie  4 , Ryo P Honda  5 , Kazuo Kuwata  6   7   8 , Mizuho Inagaki  1   9 , Tomio Yabe  1   8   9 , Izumi Nomura  1 , Tohru Suzuki  1   9
Affiliations

Capsular polysaccharide inhibits adhesion of Bifidobacterium longum 105-A to enterocyte-like Caco-2 cells and phagocytosis by macrophages

Amin Tahoun et al. Gut Pathog. .

Abstract

Background: Bifidobacterium 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.

Results: A 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.

Conclusions: Our 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.

Keywords: Bifidobacterium longum 105-A; Caco-2 cell line; Capsular polysaccharides; Cell adhesion; Phagocytosis; RAW 264.7.

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Figures

Fig. 1
Fig. 1
CPS/EPS gene cluster of B. longum 105-A. The gene cluster from BL105A_0403 to BL105A_0427, which located nucleotide number 476,499–509,716, in genome sequence (AP014658) was shown. The enzyme and gene names, annotated by blast homology search (Table 1), were indicated. Putative operons, eps1-4, were shown with arrows
Fig. 2
Fig. 2
Light microscope images of B. longum 105-A and ∆cpsD mutant. India ink staining, the polysaccharide capsule appears as a clear halo around the microorganism in the wild-type B. longum 105-A (a), while this layer not present in its ∆cpsD mutant (b). TEM images with negative staining. Wild-type expressing ca. 0.2-µm-thick CPS layer (c, and white arrow), while the mutant did not possess the CPS but expressing long and dense fimbriae (d, and black arrow). White bars 0.2 µm
Fig. 3
Fig. 3
qPCR to confirm the deletion of cpsD and its influence on downstream genes. Relative expression of wild-type B. longum 105-A (white) against B. longum 105-A ∆cpsD (black) of BL105A_0405 (cpsD), BL105A_0406, BL105A_0408, BL105A_0414, and BL105A_0424 (as indicated numbers) were analyzed by qPCR using the 2 −∆∆CT method. The rnaP gene was used as housekeeping control to normalize the data. In comparison to the wild-type B. longum 105-A, the expression of BL105A_0405 (cpsD) gene was undetectable in the ΔcpsD mutant. The downstream gene BL105A_0406, was significantly decreased by approximately 0.46% (P < 0.001) in the ΔcpsD mutant. The expression of genes, BL105A_0408, BL105A_0414, and BL105A_0424, were also decreased to 20% (P < 0.01). Temporal operon name also indicated below. ND not detected
Fig. 4
Fig. 4
Effects CPS on the bacterial resistance to bile salts and acid. a Survival rate of B. longum 105-A (white) and B. longum 105-A ∆cpsD (black) in MRS broth, which is adjusted to indicated pH. b Adaptation by low pH. Cells were pre-incubated at 37 °C, for 2 h in MRS (pH 6.5; white) or pH 4.5 (black), then transferred to the same medium but pH 3.5 incubate for 2 h. B. longum 105-A (left) and B. longum 105-A ∆cpsD (right). c Survival rate of B. longum 105-A (white) and B. longum 105-A ∆cpsD (black) in MRS broth contained bile acid, 0–0.3% (w/v)
Fig. 5
Fig. 5
Bifidobacterium longum 105-A adherence to Caco-2 cells and phagocytosis by murine macrophage. 70% confluent monolayers of Caco-2 cells were challenged with B. longum 105-A (a) and its ∆cpsD mutant (b) at MOI = 100, then determined by phase contrast microscopy. No adherent bacterial cell was observed in the wild-type (a) but a lot of adherent bacteria were observed in ∆cpsD mutant (b). The number of attached ∆cpsD cells per Caco-2 cell was 7.8 ± 2.3 (nucleus ± SD). Three slides for each bacterial strain and at least 20 fields per slide were counted. B. longum 105-A phagocytosis by murine macrophage. Semi-confluent RAW 264.7 murine macrophage was challenged with B. longum 105-A (c) and its ∆cpsD mutant (d) for 30 min. Then, the medium was removed and the cells were washed 5 times with PBS and replace to DMEM containing gentamycin (100 µg/ml) and incubate for another 1 h. The coverslips were then washed 3 times with PBS and the cells were fixed with methanol and stained with Giemsa stain. In the wild-type, no bacterial cell was observed both inside and outside of macrophage cell (c) but ∆cpsD mutant was internalized into Raw 264.7 murine macrophage cells (d). The number of internalized bacterial ce1ls per macrophage cell was 4.1 ± 1
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References

    1. Walker AW, Lawley TD. Therapeutic modulation of intestinal dysbiosis. Pharmacol Res. 2013;69:75–86. doi: 10.1016/j.phrs.2012.09.008. - DOI - PubMed
    1. Scholtens PAMJ, Oozeer R, Martin R, Amor K Ben, Knol J. The early settlers: Intestinal microbiology in early life. Annu Rev Food Sci Technol. 2012;3:425–47. http://www.annualreviews.org/doi/abs/10.1146/annurev-food-022811-101120. - DOI - PubMed
    1. Lane JA, Mehra RK, Carrington SD, Hickey RM. The food glycome: a source of protection against pathogen colonization in the gastrointestinal tract. Int J Food Microbiol. 2010;142:1–13. doi: 10.1016/j.ijfoodmicro.2010.05.027. - DOI - PubMed
    1. Mitsuoka T. Establishment of intestinal bacteriology. Biosci Microbiota Food Health. 2014;33:99–116. doi: 10.12938/bmfh.33.99. - DOI - PMC - PubMed
    1. Jandhyala SM. Role of the normal gut microbiota. World J Gastroenterol. 2015;21:8787. doi: 10.3748/wjg.v21.i29.8787. - DOI - PMC - PubMed