Lactobacillus rhamnosus GG Outcompetes Enterococcus faecium via Mucus-Binding Pili: Evidence for a Novel and Heterospecific Probiotic Mechanism

Abstract

Vancomycin-resistant enterococci (VRE) have become a major nosocomial threat. Enterococcus faecium is of special concern, as it can easily acquire new antibiotic resistances and is an excellent colonizer of the human intestinal tract. Several clinical studies have explored the potential use of beneficial bacteria to weed out opportunistic pathogens. Specifically, the widely studied Lactobacillus rhamnosus strain GG has been applied successfully in the context of VRE infections. Here, we provide new insight into the molecular mechanism underlying the effects of this model probiotic on VRE decolonization. Both clinical VRE isolates and L. rhamnosus GG express pili on their cell walls, which are the key modulators of their highly efficient colonization of the intestinal mucosa. We found that one of the VRE pilus clusters shares considerable sequence similarity with the SpaCBA-SrtC1 pilus cluster of L. rhamnosus GG. Remarkable immunological and functional similarities were discovered between the mucus-binding pili of L. rhamnosus GG and those of the clinical E. faecium strain E1165, which was characterized at the genome level. Moreover, E. faecium strain E1165 bound efficiently to mucus, which may be prevented by the presence of the mucus-binding SpaC protein or antibodies against L. rhamnosus GG or SpaC. These results present experimental support for a novel probiotic mechanism, in which the mucus-binding pili of L. rhamnosus GG prevent the binding of a potential pathogen to the host. Hence, we provide a molecular basis for the further exploitation of L. rhamnosus GG and its pilins for prophylaxis and treatment of VRE infections.

Importance: Concern about vancomycin-resistant Enterococcus faecium causing nosocomial infections is rising globally. The arsenal of antibiotic strategies to treat these infections is nearly exhausted, and hence, new treatment strategies are urgently needed. Here, we provide molecular evidence to underpin reports of the successful clinical application of Lactobacillus rhamnosus GG in VRE decolonization strategies. Our results provide support for a new molecular mechanism, in which probiotics can perform competitive exclusion and possibly immune interaction. Moreover, we spur further exploration of the potential of intact L. rhamnosus GG and purified SpaC pilin as prophylactic and curative agents of the VRE carrier state.

FIG 1

Sequence homology of pilus gene clusters of Enterococcus faecium E1165 and Lactobacillus rhamnosus GG. Pilus gene clusters of both E. faecium E1165 (∼99% similar to the TX16 type strain) (25) and L. rhamnosus GG (23) are shown. Overall, the clusters have a similar buildup and share a high sequence homology (>50% similarity), and both harbor a sortase (black), a tip adhesin (white), and two pilins (gray). The tip pilins of both strains ORF 2571 (TX16 and E1165) and spaC share 33% identity (49% positive matches). Arrow sizes reflect gene sizes.

FIG 2

L. rhamnosus GG SpaC pilin-specific antibodies bind to E1165 pili. Immunogold labeling microscopy using SpaC antibodies shows binding of these L. rhamnosus GG major pilin-specific antibodies to the pili of E. faecium E1165 cells. Representative experimental results are shown (scale bar, 200 nm).

FIG 3

L. rhamnosus GG SpaC pilin antibodies colocalize with E. faecium PilB pilin antibodies on E1165 pili. E. faecium E1165 cells were incubated with gold particles either labeled with PilB antibodies (10-nm particles, black arrows in C) or SpaC antibodies (15-nm particles, white arrows in C). Both labels colocalize on the pili of E1165, indicating their specificity for the PilB-type pili of E. faecium E1165 expressed by PGC-3. (A) Complete E1165 cells (scale bar, 200 nm). (B and C) Zoomed-in image of the labeled pili (scale bars, respectively, 100 nm and 50 nm). Results of a representative experiment are shown.

FIG 4

Serum from VRE-colonized patients interacts with L. rhamnosus GG pili, as shown by immunogold labeling microscopy of L. rhamnosus GG in patient serum samples. (A and B) Serum samples from two different patients infected with VRE. (C) Serum from a healthy patient (scale bar, 200 nm). Pictures of representative experiments are shown.

FIG 5

L. rhamnosus GG-specific antibodies and purified SpaC pilin interfere with the mucus-binding capacity of E. faecium E1165. The high adhesion capacity of L. rhamnosus GG to mucus can be explained by the presence of its SpaCBA pili, as adhesion of PB12, the mutant lacking pili, significantly drops (P < 0.0001). E. faecium E1165 can adhere well to mucus. Antibodies raised against the SpaC pilin of L. rhamnosus GG significantly reduce binding of E. faecium E1165 to mucus (P < 0.0001), as do antibodies raised against complete L. rhamnosus GG cells (P = 0.0004). The mucus-binding SpaC pilin purified from E. coli may also significantly interfere with binding of E1165 to mucus (P = 0.0011). Error bars depict the standard error of the mean (SEM) and data represent findings from at least triplicate experiments.