Efficient utilization of complex N-linked glycans is a selective advantage for Bacteroides fragilis in extraintestinal infections

Abstract

Bacteroides fragilis is the most common anaerobe isolated from clinical infections, and in this report we demonstrate a characteristic of the species that is critical to their success as an opportunistic pathogen. Among the Bacteroides spp. in the gut, B. fragilis has the unique ability of efficiently harvesting complex N-linked glycans from the glycoproteins common to serum and serous fluid. This activity is mediated by an outer membrane protein complex designated as Don. Using the abundant serum glycoprotein transferrin as a model, it has been shown that B. fragilis alone can rapidly and efficiently deglycosylate this protein in vitro and that transferrin glycans can provide the sole source of carbon and energy for growth in defined media. We then showed that transferrin deglycosylation occurs in vivo when B. fragilis is propagated in the rat tissue cage model of extraintestinal growth, and that this ability provides a competitive advantage in vivo over strains lacking the don locus.

Fig. 1.

In vivo PUL gene expression during growth in the rat tissue cage model. Samples for analyses were pooled from five animals 1, 4, and 8 d postinoculation. (A) Expression of donC in samples from the rat tissue cage relative to midlogarithmic cultures grown in DM–glucose. Expression was measured in triplicate samples by qRT-PCR and normalized to the amount of 16S rRNA. Values are shown with SDs. (B) Induction of SusC homologs during growth in vivo relative to DM–glucose. Shown are the highly induced in vivo susC-like genes associated with PULs in cluster 1. Clusters were determined from expression microarray data analyzed by k-means clustering using the standard Pearson’s correlation coefficient distance metric. All induction values were significant at P < 0.01.

Fig. 2.

Induction of donC expression during growth on mucin glycans. Strain BF638R was grown in DM containing 2% porcine gastric mucin glycans as the sole carbon/energy source. The induction of donC relative to growth on glucose was determined by qRT-PCR. The results are overlaid on a typical growth curve with mucin glycans (OD₅₅₀, gray diamonds). The qRT-PCR results represent two independent experiments (gray squares) with SDs shown.

Fig. 3.

Deglycosylation of transferrin is mediated by the Don PUL. (A) Serous fluid obtained from uninfected tissues cages was incubated for 3 h with PBS or B. fragilis cells induced by growth in DM–mucin glycan medium. Samples were analyzed by 12% SDS/PAGE and stained with Ponceau S. The arrow indicates the location of transferrin above the abundant serum albumin protein. (B) Deglycosylation analysis of human transferrin. Human transferrin was incubated with WT or ∆don cells induced by growth in DM–mucin glycan medium. Samples were analyzed by SDS/PAGE and stained for protein with Coomassie blue (Co) or SNA lectin to detect N-linked glycans as described in Materials and Methods.

Fig. 4.

B. fragilis can grow with transferrin as the sole source of carbon/energy. Growth curves are shown for WT or ∆don strains in DM with different carbon/energy sources. An overnight inoculum of 2% was used, and the OD₅₅₀ was measured at specific time intervals. Dashed lines represent ∆don and solid lines represent WT. Squares represent glucose (0.4%), triangles human transferrin (25 mg/mL), and circles BSA (25 mg/mL). Each growth curve represents two biological repeats with the SDs shown.

Fig. 5.

Growth in vivo is enhanced by deglycosylation of transferrin. (A) Growth curve for WT (circles) and ∆don (squares) strains inoculated separately into rat tissue cages. (B) Mixed culture competition assay. Mixtures of WT (stippled bars) and ∆don (cross-hatched bars) cells were prepared in a 1:1 ratio, inoculated into five rats, and the cfu per milliliter determined. The percentage of WT or ∆don cells was determined by screening the total cell counts for tetracycline-resistant colonies. (C) Coomassie blue- stained SDS/PAGE gel of serous fluid samples obtained following growth of strains in the rat tissue cage. Samples from the WT and ∆don (∆) strains were compared with uninoculated controls (un). The arrowhead indicates the migration of the glycosylated transferrin, and red squares indicate deglycosylated transferrin. The results in A and B are averaged from two biological repeats with the SDs shown, and C shows a representative result.

Fig. 6.

Deglycosylation of human transferrin by medically important Bacteroides species. Pure human transferrin was used in standard 3-h deglycosylation assays with midlogarithmic phase cells of Bacteroides species grown in DM–mucin glycan media. Samples were analyzed by SDS/PAGE with Coomassie blue (CO) staining and on duplicate SDS/PAGE gels, followed by SNA glycan staining. Lanes: 1, PBS; 2, B. fragilis (638R); 3, B. fragilis (American Type Culture Collection 25285); 4, B. thetaiotaomicron (IB116); 5, Bacteroides uniformis; 6, Bacteroides ovatus; 7, B. vulgatus; 8, Parabacteroides distasonis; 9, Parabacteroides merdae.