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

Abstract

In spite of the wealth of clinical evidence supporting the health benefits of Lactobacillus rhamnosus GG in humans, there is still a lack of understanding of the molecular mechanisms behind its probiosis. Current knowledge suggests that the health-promoting effects of this probiotic strain might be partly dependent on its persistence in the intestine and adhesion to mucosal surfaces. Moreover, L. rhamnosus GG contains mucusbinding pili that might also explain the occupation of its ecological niche as a comparatively less stringent allochthonous intestine-dwelling bacterium. To uncover additional surface proteins involved in mucosal adhesion, we investigated the adherence properties of the only predicted protein (LGG_02337) in L. rhamnosus GG that exhibits homology with a known mucus-binding domain. We cloned a recombinant form of the gene for this putative mucus adhesin and established that the purified protein readily adheres to human intestinal mucus. We also showed that this mucus adhesin is visibly distributed throughout the cell surface and participates in the adhesive interaction between L. rhamnosus GG and mucus, although less prominently than the mucus-binding pili in this strain. Based on primary structural comparisons, we concluded that the current annotation of the LGG_02337 protein likely does not accurately reflect its predicted properties, and we propose that this mucus-specific adhesin be called the mucus-binding factor (MBF). Finally, we interpret our results to mean that L. rhamnosus GG MBF, as an active mucus-specific surface adhesin with a presumed ancillary involvement in pilus-mediated mucosal adhesion, plays a part in the adherent mechanisms during intestinal colonization by this probiotic.The commensal Gram-positive lactobacilli are one of the first groups of bacteria to inhabit the human gastrointestinal (GI) tract (16) and include some strains (autochthonous) that colonize the intestine stably throughout the lifetime of the host (31). In addition, there are those strains, called allochthonous, that persist only briefly in the intestine, many of these being probiotics (1, 9, 13) and understood to stimulate health-benefiting immune responses in host intestinal cells (for a review, see reference 14) or cause competitive displacement of invading pathogens (for a review, see reference 35). 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. (3,7,8,11,33) has identified the presence of various-sized putative mucus ...

Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells

“…We have previously observed that the large-sized surface protein MabA can also modulate binding to Caco-2 cells (40) and that the recombinant Mbf protein can also bind to human mucus (41), but the mutant analysis in this study showed that their overall role in L. rhamnosus GG adhesion to IECs is a minor one compared to that of the SpaCBA pilus. However, a combination of different surface-localized proteins for host cell binding in L. rhamnosus GG agrees with the proposed "zipper model" for pilusmediated adhesion among Gram-positive pathogens, in which initial bacterial contact with host cells is made with the long and adhesive pilus fibers, followed by other surface adhesins helping the formation of an intimate zone of contact between the bacterial and host cells (37).…”

“…In previous studies, wild-type L. rhamnosus GG has been shown to display a high adhesion capacity to IECs and mucus (reviewed in reference 8). Recently, the SpaC pilin tip adhesin and mucus binding factor Mbf of L. rhamnosus GG were found to be involved in adhesion to human mucus (19,41). In this study, we aimed to identify key adhesins of L. rhamnosus GG for binding to Caco-2 IECs based on a comparative knockout mutant analysis.…”

In vitro characterization of aggregation and adhesion properties of viable and heat-killed forms of two probiotic Lactobacillus strains and interaction with foodborne zoonotic bacteria, especially Campylobacter jejuni

“…The reduction of adhesion can be explained by the heatsensitive proteinaceous nature of the molecules involved in Lb. rhamnosus GG adhesion (von Ossowski et al, 2010(von Ossowski et al, , 2011. Interestingly, heat-killed forms of both Lb.…”

“…farciminis CIP 103136 (Mercier-Bonin et al, 2011) and probiotic reference strain Lb. rhamnosus GG (Kirjavainen et al, 1998;von Ossowski et al, 2011;Van den Abbeele et al, 2012). Mucin is a glycosylated protein and attachment of bacteria to mucin involves specific bacterial proteins such as mucus-binding proteins and other lectins or adhesins.…”

“…farciminis CIP 103136 (Mercier-Bonin et al, 2011) and Lb. rhamnosus GG, known as mucusbinding strains (von Ossowski et al, 2010(von Ossowski et al, , 2011. Compared to the reference strains, the two tested lactobacilli showed lower ability to adhere to mucin, especially for Lb.…”