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.

Fig. 1.

The colonization properties of L. rhamnosus GG and LC705 in the human GI tract. A short-term intervention study involving 12 healthy adults assessed the colonization properties of two L. rhamnosus strains in the GI tract. Fecal samples were collected during the intervention (−7 days) and postintervention (0–21 days) periods and analyzed for the presence of strains GG (black) and LC705 (white). The fraction of subjects containing the L. rhamnosus strains above a detection threshold is presented (refer to SI Text for further details.)

Fig. 2.

The unique spaCBA pili cluster of L. rhamnosus GG and its expression. (A) Schematic illustration of the spaCBA and spaFED gene clusters. Depicted are the sortase (dark gray arrows), pilin subunits (white arrows), Cna protein B-type domain (gray), E box (black), YPKN pilin-like motif (black diagonal stripes), LPXTG-like motif (light gray arrowheads), and secretion signal (light gray). The von Willebrand type A domain is indicated by black and gray vertical stripes. The best homology match between proteins (gray arrows) and % amino-acid identity are indicated. (B) Detection of cell wall-associated proteins by immunoblotting. E. coli-purified SpaC (lane 1), cell wall protein extracts of strains GG-ΩspaC (lane 2), GG (lane 3), and LC705 (lane 4) were prepared from cells grown in Man–Rogosa–Sharpe broth, separated by SDS/PAGE, electroblotted to a membrane, and probed with SpaC antiserum. The positions of the monomeric SpaC protein (*), HMW protein complexes, and molecular weight standards (kDa) are indicated.

Fig. 3.

Identification of pili in L. rhamnosus GG by immunogold electron microscopy. L. rhamnosus GG was grown to stationary phase, treated with anti-SpaC serum, labeled with protein A-conjugated gold particles (10 nm), negatively stained, and examined by transmission electron microscopy. (A) High-resolution electron micrograph showing multiple pili and an isometric bacteriophage (black arrow). Also included is a panel inset adjusted for heightened contrast and darkness to highlight the pilus ultrastructure (white arrow). (B) Electron micrograph showing pili clustered at the cell poles. (Bars: A, 200-nm; B, 500-nm.)

Fig. 4.

In vitro competitive binding of L. rhamnosus GG SpaC pilin to human intestinal mucus. Recombinant SpaC pilin was radiolabeled (¹²⁵I), and the binding to human intestinal mucus was competitively inhibited by 2-, 10-, and 100-fold increases in unlabeled SpaC protein (in molar amounts). Binding by radiolabeled ovalbumin defined the background level of mucus binding. Binding results are an average of 4 to 6 measurements (standard deviations are depicted), and all dataset comparisons were considered significant (P = 0.05). Competition with unlabeled SpaC caused dose-related inhibition of mucus binding (P = 2.0 × 10⁻⁷).

Fig. 5.

Adhesion of L. rhamnosus GG and GG-ΩspaC to human intestinal mucus. Radiolabeled (³H) cells of the L. rhamnosus GG strain (with or without SpaC antiserum pretreatment) and the GG-ΩspaC mutant were tested for binding to human intestinal mucus. Cell-binding results are averaged from 4 to 6 measurements, and the standard deviations are depicted. Comparisons between strain GG in the absence or presence of SpaC antiserum and between strain GG and the GG-ΩspaC mutant were considered significant (P = 0.05).