Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein

Abstract

To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins (spaCBA) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.genome ͉ probiotics ͉ adhesion ͉ pilus ͉ lactic acid bacteria

Functional Characterization of a Mucus-Specific LPXTG Surface Adhesin from Probiotic Lactobacillus rhamnosus GG

“…Thus far, the precise molecular mechanisms that differentiate the colonization ability between autochthonous and allochthonous intestinal lactobacilli remain undefined (42), although they are likely to be partly dependent on a diverse range of cell surface adhesion molecule-mediated interactions with the host intestinal mucosa. With that being said, there are a growing number of reports in the literature that indicate that lactobacillar adherence to the intestinal mucosal layer is mediated by surface proteins with a mucus-binding capacity (15,21,22,25,30,32,33,41). Moreover, homology-driven genome mining in several Lactobacillus spp.…”

“…In a recent study, we reported that proteinaceous pilus-like structures protrude from the cell surface of a widely used probiotic Lactobacillus strain (21). Earlier studies have primarily characterized Gram-positive pili as virulence factors in pathogen-mediated disease and illness (for a review, see reference 37).…”

“…Earlier studies have primarily characterized Gram-positive pili as virulence factors in pathogen-mediated disease and illness (for a review, see reference 37). However, with the discovery that Lactobacillus rhamnosus GG is a piliated strain (21) and that an intestinal mucus-binding capacity is associated with the corresponding pili (called SpaCBA) (21,41), new light has been shed on the putative role of piliation as a novel surface-localized feature in mediating intestinal colonization by probiotic lactobacilli. As a typical probiotic, L. rhamnosus GG adopts the characteristic colonization behavior associated with allochthonous bacteria (42) and so persists only transiently in the GI tract (1).…”

“…As a typical probiotic, L. rhamnosus GG adopts the characteristic colonization behavior associated with allochthonous bacteria (42) and so persists only transiently in the GI tract (1). However, compared to the genetically similar but non-SpaCBApiliated L. rhamnosus LC705 strain, piliation might offer a possible explanation for the heightened ability of L. rhamnosus GG to adhere to intestinal mucosal surfaces (39) and to occupy its intestinal niche with greater duration (21). In this context, L. rhamnosus GG might be regarded as a more sustainable allochthonous strain than L. rhamnosus LC705.…”

“…Up to now, SpaCBA pilus fibers have been characterized as the predominant surface-localized component for L. rhamnosus GG mucosal adhesion (21,41). However, there are growing indications that other LPXTG-anchored cell wall proteins, such as MabA (29), also function in the adherence of this probiotic strain to the host intestinal mucosa.…”