Role of glycan synthesis in colonization of the mammalian gut by the bacterial symbiont Bacteroides fragilis

Abstract

Bacteroides species are the most abundant Gram-negative bacteria of the human colonic microbiota. These endogenous organisms are unique in that they synthesize an extensive number of phase-variable surface polysaccharides. Pathogenic bacteria phase vary expression of surface molecules for immune evasion, but the importance of the synthesis of multiple phase-variable polysaccharides to these commensal bacteria is unknown. We previously showed that a Bacteroides fragilis mutant unable to synthesize 4 of the 8 capsular polysaccharides and unable to glycosylate proteins properly is rapidly outcompeted by the wild-type strain for colonization of the gnotobiotic mouse intestine. In the present study, we constructed mutants defective only in capsule polysaccharide synthesis to define better the importance of these surface molecules to intestinal colonization. We discovered a key enzymatic activity required for synthesis of 7 of the 8 capsular polysaccharides. Deletion of its gene resulted in the first B. fragilis mutant able to synthesize only one phase-variable polysaccharide, and further mutation resulted in a stable acapsular mutant. We show that the acapsular mutant is rapidly outcompeted, but synthesis of a single polysaccharide is sufficient for the organism to colonize the gnotobiotic intestine competitively. These data demonstrate that initial colonization of the gnotobiotic mouse intestine by B. fragilis requires that the organism synthesize only a single polysaccharide and suggest that the synthesis of multiple phase-variable polysaccharides is important for the bacteria's long-term maintenance in the normally complex and competitive ecosystem.

Fig. 1.

Functional and genetic analyses of ungD1 and ungD2. (A) Schematic diagram of the B. fragilis 9343 chromosome with the locations of the 8 capsule loci (PSA–PSH), ungD1, and ungD2. (B) Ability of ungD1 and ungD2 to complement a P. aeruginosa wbpM mutant. Lane 1: SeeBlue Plus2 prestained protein ladder (Invitrogen). Lane 2: P. aeruginosa PAO1. Lane 3: P. aeruginosa PAO1 wbpM::Gm. Lane 4: P. aeruginosa PAO1 wbpM::Gm pUCP18 (vector control). Lane 5: P. aeruginosa PAO1 wbpM::Gm pLEC216 (ungD1). Lane 6: P. aeruginosa PAO1 wbpM::Gm pLEC217 (ungD2). (C) Production of each of the 8 capsular polysaccharides by wild type, ΔungD1, ΔungD2, and ΔungD1ΔungD2 mutants and mutants with ungD1 and ungD2 in trans and by the acapsular mutant (ΔungD1ΔungD2ΔPSH). (D) Whole-cell lysates of B. fragilis mutants probed with antiserum generated to wild-type B. fragilis. Lane 5 is blank. The ΔungD1ΔungD2ΔPSH in vivo sample is bacteria isolated from the feces of mice monoassociated for 3 days and passaged in vitro once before analysis. (E) Growth comparison of B. fragilis 9343 wild type, ΔungD1ΔungD2, and ΔungD1ΔungD2ΔPSH in supplemented basal medium. The average absorbance at 600 nm of 5 replicate cultures is shown.

Fig. 2.

Analysis of glycoprotein synthesis by ungD mutants. (A) Phosphoimager scan of the blot of a PAGE gel of whole-cell lysates of B. fragilis mutants grown in the presence of ³H-l-fucose. (B) Glycostain analysis of whole-cell lysates separated by PAGE.

Fig. 3.

Phase variation of PSH in ΔungD1ΔungD2 mutant. (A) Appearance of cultures after overnight growth demonstrating that most of the bacteria with deletions of ungD2 are settled at the bottom of the tube compared with the wild-type culture and that the acapsular mutant grows as a completely aggregated population. (B) PCR-digestion scheme used to determine quantitatively the portions of a given population with the PSH promoter in the on and off orientations. (C) PCR-digestion results for the PSH promoter from the aggregated bacteria versus those growing in suspension for the ΔungD1ΔungD2 mutant. (D) PSH promoter orientations of bacteria from fecal samples of mice monoassociated with ΔungD1ΔungD2 for various intervals. (E) PSH promoter orientations of bacteria from fecal samples of 2 mice monoassociated with wild type for 7 or 28 days.