Structural basis for adaptation of lactobacilli to gastrointestinal mucus
Summary The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the underpinning mechanisms of these interactions are not understood. Here, we provide structural and functional insights into the c...
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| Veröffentlicht in: | Environmental microbiology Jg. 16; H. 3; S. 888 - 903 |
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| Hauptverfasser: | , , , , , , , |
| Format: | Journal Article |
| Sprache: | Englisch |
| Veröffentlicht: |
Oxford
Blackwell Publishing Ltd
01.03.2014
Blackwell Wiley Subscription Services, Inc |
| Schlagworte: | |
| ISSN: | 1462-2912, 1462-2920, 1462-2920 |
| Online-Zugang: | Volltext |
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| Zusammenfassung: | Summary
The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the underpinning mechanisms of these interactions are not understood. Here, we provide structural and functional insights into the canonical mucus‐binding protein (MUB), a multi‐repeat cell‐surface adhesin found in Lactobacillus inhabitants of the GI tract. X‐ray crystallography together with small‐angle X‐ray scattering demonstrated a ‘beads on a string’ arrangement of repeats, generating 174 nm long protein fibrils, as shown by atomic force microscopy. Each repeat consists of tandemly arranged Ig‐ and mucin‐binding protein (MucBP) modules. The binding of full‐length MUB was confined to mucus via multiple interactions involving terminal sialylated mucin glycans. While individual MUB domains showed structural similarity to fimbrial proteins from Gram‐positive pathogens, the particular organization of MUB provides a structural explanation for the mechanisms in which lactobacilli have adapted to their host niche by maximizing interactions with the mucus receptors, potentiating the retention of bacteria within the mucus layer. Together, this study reveals functional and structural features which may affect tropism of microbes across mucus and along the GI tract, providing unique insights into the mechanisms adopted by commensals and probiotics to adapt to the mucosal environment. |
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| Bibliographie: | BBSRC Institute Strategic Programme - No. BB/J004529/1; No. BB/K019554/1 ark:/67375/WNG-QJ5BTQGV-8 istex:6EA8C62DF2DDB9E04B05F2EDBC591C0289BDF773 ArticleID:EMI12377 Fig. S1. Secondary structure determination of Mub-proteins by circular dichroism (CD). (A) Far UV CD spectra of purified Mub-repeat and Nterm proteins collected over scan range 180-260 nm [see also (MacKenzie et al., 2009)]. (B) The proportion of secondary structural elements was calculated using the CONTIN analysis program, revealing a 64-71% content of β-sheet and β-turns and a low percentage of α-helices for all tested Mub-proteins. Nterm showed an α-helix content of 6.2% and a 56.2% content of β-sheet and β-turns.Fig. S2. SAXS analysis of single Mub-repeat proteins and solution shape reconstruction. SAXS data for the type 1 Mub-RV (blue) and Mub-RI (red) repeats and the type 2 Mub-R5 repeat (green) are presented. (A) The experimental scattering curves are shown as the logarithm of the scattering intensity I (black dots) as a function of the reverse momentum transfer s and presented offset for better visualisation. Overlaying the scattering profiles, are fits of the reconstructed averaged models for Mub-RV, Mub-RI and Mub-R5 generated by GASPOR. (B) Pair distribution functions P(r) were generated from the experimental scattering using GNOM. Low resolution shape reconstructions of Mub-RV (C), Mub-RI (D) and Mub-R5 (E) were achieved by GASPOR and high resolution structures of Mub-RV (blue) and Mub-R5 (green) were manually docked and refined using Sculptor and SITUS.Fig. S3. Purification of native full-length MUB protein. Native MUB protein was purified from Lactobacillus reuteri ATCC 53608 spent media in a multi-step process. (A) In a final size exclusion chromatography (SEC) MUB elutes with the void volume of the column in elution fractions of high protein homogeneity verified by SDS-PAGE gel (B) and specific protein detection via anti-Mub-R5 and -Mub-RI antibodies after Western blotting (C) 1: cell pellet, 2: extracted protein before SEC, 3-11: MUB-containing elution fractions (red box).Fig. S4. MUB protein binding to HT29-MTX cell monolayers. HT29-MTX cell monolayers (n = 12) were incubated with MUB for 2 h at 37°C, followed by staining with rabbit anti-Mub-R5 and goat anti-rabbit Alexa Fluor 488 secondary antibody (A). HT29-MTX cell monolayers were stained with polyclonal anti-MUC5AC followed by goat anti-rabbit Alexa Fluor 488 secondary antibody (B). Bright field images are shown adjacent to fluorescent images to demonstrate the correlation of fluorescence staining with mucus droplet structures. Magnification ×400; scale bars, 50 μm.Table S1. Size of Mub repeats and the N-terminal domain. Table S2. Mub-RV data collection and refinement parameters. Table S3. SAXS data statistics. Table S4. Frequencies of Mub domain pairs in close association. Table S5. Predicted protein sequences with at least 8 Mub domains. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 |
| ISSN: | 1462-2912 1462-2920 1462-2920 |
| DOI: | 10.1111/1462-2920.12377 |