Structural basis for adaptation of lactobacilli to gastrointestinal mucus

Abstract

SummaryThe 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 cellsurface 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.

Use of Atomic Force Microscopy to Study the Multi-Modular Interaction of Bacterial Adhesins to Mucins

“…MUB is a large, modular, cell surface protein (~350 kDa) made up of 14 Mub repeats of ~20 kDa, divided in two types (Mub1 and Mub2) based on sequence identity and an N-terminal domain of unknown function [24,25]. Small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) demonstrated a “beads on a string” arrangement of the Mub repeats, generating ~174 nm long protein fibrils [26]. The binding of the full-length MUB to mucus appears to be mediated via multiple interactions involving terminal sialylated mucin glycans, as shown by the net reduction in MUB adhesion to (1) mucin-secreting epithelial cells grown in the presence of an inhibitor of sialylation; or to (2) mammalian intestinal tissue after chemical desialylation [26].…”

“…Small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) demonstrated a “beads on a string” arrangement of the Mub repeats, generating ~174 nm long protein fibrils [26]. The binding of the full-length MUB to mucus appears to be mediated via multiple interactions involving terminal sialylated mucin glycans, as shown by the net reduction in MUB adhesion to (1) mucin-secreting epithelial cells grown in the presence of an inhibitor of sialylation; or to (2) mammalian intestinal tissue after chemical desialylation [26]. However, direct measurements of MUB binding to mucin glycans are lacking.…”

“…Methodologies to screen for bacterial adhesion to mucins have previously employed thin layer chromatography overlay [26], enzyme-linked immunosorbent assay [15], micro-titre plate assays [8,25,27], surface plasmon resonance [16,28,29,30,31], fluorescence spectroscopy [20], mucin microarrays [32], flow cytometry [33], and cell-based assays [26,34,35,36]. However, due to the complexity and diversity of mucin glycosylation, these methods typically provide qualitative binding data indicating only presence or absence of interaction.…”

Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins

“…The MucBD-containing proteins have been predominantly identified in lactobacilli that are naturally found in intestinal niches 16) and promote cell adhesion to mucins. [17][18][19] However, the cell wall surface anchor-containing MBF adhesion properties to intestinal mucosa-associated components, including ECM proteins, are poorly studied in L. rhamnosus. We previously described the properties of 25 potential probiotic L. rhamnosus strains isolated from traditional Indonesian foods, including fermented Sumbawa mare milk (FSMM) produced on Sumbawa Island.…”

Safety and Antioxidant Properties of Five Probiotic <i>Lactobacillus plantarum</i> Strains Isolated from the Digestive Tract of Honey Bees

“…Adhesion to intestinal mucus is a wanted attribute for probiotic bacteria, as it increases persistence in the GIT (Gastrointestinal Tract) and the aptitude to colonize intestine efficiently [16,17]. In vitro assay facilitates a preliminary selection of strains with probiotic potential, focusing on significant features as adhesion and safety properties, antibiotics susceptibility.…”