Mucosal Adhesion Properties of the ProbioticLactobacillus rhamnosusGG SpaCBA and SpaFED Pilin Subunits

Abstract

Lactobacillus rhamnosus GG is a well-established Gram-positive probiotic strain, whose health-benefiting properties are dependent in part on prolonged residence in the gastrointestinal tract and are likely dictated by adherence to the intestinal mucosa. Previously, we identified two pilus gene clusters (spaCBA and spaFED) in the genome of this probiotic bacterium, each of which contained the predicted genes for three pilin subunits and a single sortase. We also confirmed the presence of SpaCBA pili on the cell surface and attributed an intestinal mucus-binding capacity to one of the pilin subunits (SpaC). Here, we report cloning of the remaining pilin genes (spaA, spaB, spaD, spaE, and spaF) in Escherichia coli, production and purification of the recombinant proteins, and assessment of the adherence of these proteins to human intestinal mucus. Our findings indicate that the SpaB and SpaF pilin subunits also exhibit substantial binding to mucus, which can be inhibited competitively in a dose-related manner. Moreover, the binding between the SpaB pilin subunit and the mucosal substrate appears to operate through electrostatic contacts and is not related to a recognized mucus-binding domain. We conclude from these results that it is conceivable that two pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber play a role in binding to intestinal mucus, but for the uncharacterized and putative SpaFED pilus fiber only a single pilin subunit (SpaF) is potentially responsible for adhesion to mucus.The human intestinal microbiota is comprised of more than 1,000 species of commensal and probiotic bacteria, including several members of the Gram-positive genus Lactobacillus (42, 52). Many strains of lactobacilli have a variety of health-promoting effects in humans and consequently have been used commercially as probiotics in foods and nutritional supplements (for a review, see reference 48). Often a necessary precondition for colonization of the human gastrointestinal (GI) tract by probiotic bacteria is preferential adherence to the intestinal mucosa, which in turn prolongs and stabilizes intestinal residence, possibly triggering a variety of defensive host cell immune responses and excluding pathogenic bacteria by competitive inhibition or steric hindrance (48). The outermost layer of the intestinal mucosa, which is a secreted and hydrated mucus gel that acts as a protective barrier and filter, consists primarily of a heterogeneous mixture of highly glycosylated membrane-associated and secreted glycoproteins called mucins (36). Although many studies have demonstrated that various probiotic Lactobacillus spp. adhere initially to the mucus gel layer, relatively few details about the overall molecular mechanism of mucosal adhesion are known (for a review, see reference 23). Nonetheless, several studies have reported that the adherence of Lactobacillus cells to the mucosal barrier is frequently due to a surface protein-mediated interaction. For example, Rojas et al. (44) determined that the ability of Lactobacillus fermentum...

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

“…Recombinant MBF protein production and purification was performed using the procedure essentially as described previously (21,41). Briefly, E. coli cells harboring the pKTH5323 plasmid were grown until mid-log phase at 37°C in kanamycinsupplemented (50 g/ml) LB medium, and MBF protein production was then induced for 3 h by 1 mM isopropyl ␤-D-1-thiogalactopyranoside (IPTG).…”

“…rhamnosus GG MBF antiserum preparation. Antiserum specific for L. rhamnosus GG MBF protein was produced using the method described previously (21,41). In brief, rabbits were first injected subcutaneously with a 1-ml volume of 400 g purified recombinant MBF protein and Freund's complete adjuvant (1:1 mixture), and then 1-ml subcutaneous booster injections of 200 g MBF protein in Freund's incomplete adjuvant (1:1 mixture) were administered at 3-week intervals over a 9-week period.…”

“…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.…”

“…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.…”

“…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).…”