The Bacteroides fragilis pathogenicity island links virulence and strain competition

Abstract

The mature microbiome is a stable ecosystem that resists perturbation despite constant host exposure to exogenous microbes. However, the microbial mechanisms determining microbiome development and composition are poorly understood. We recently demonstrated that a non-toxigenic B. fragilis (NTBF) strain restricts enteric colonization by an enterotoxigenic (ETBF) strain dependent on a type VI secretion system (T6SS). We show here that a second enterotoxigenic strain is competent to colonize, dependent on the Bacteroides fragilis pathogenicity island (BFPAI). Additional data showing complex environmental regulation of the Bacteroides fragilis toxin (BFT) suggest that virulence factors may be adapted to modify the colonic niche to provide a strain-specific colonization advantage. We conclude that more complex models of host-microbe-microbiome interactions are needed to investigate this hypothesis.

Keywords: bacteroides; competition; pathogenesis; secretion; toxin.

Figure 1.

bfpai locus deletion impairs fitness during competitive secondary colonization. (A-B) Mice were primarily colonized with NTBF strain TM4000 638R and subsequently challenged with either ETBF strain ATCC 43859 or an isogenic mutant with bfpai deleted (Δbfpai). Clindamycin (100 mg/L) was maintained in drinking water throughout the experiment. Fecal CFU was monitored for 5 weeks post-challenge. N = 4 mice per group. Figures are representative of 3 independent experiments. Error bars: mean +/− s.e.m (A), mean +/− s.d. (B). Dashed lines indicate the limit of detection. n.s., not significant.

Figure 2.

BFT is transcriptionally regulated by fermentable carbohydrates. (A) Full-length protoxin (FLBFT) in cell pellet and cleaved active BFT (BFT*) in culture supernatant was probed by western blot during log (5, 6 h) or early stationary (7, 8 h) phase growth from cultures with or without 0.5% glucose. (B) RNA from cell pellet in (a) was assayed by qPCR at various time points during log (5, 6 h) or early stationary (7, 8 h) phase growth from cultures with (filled bars) or without (empty bars) 0.5% glucose. *p<0.05, ** p<0.01; ns, not significant. (C) B. fragilis cultures show log phase growth in BHIS (filled squares) with early stationary phase beginning around 7–8 hours. The addition of 0.5% glucose to BHIS (open squares) does not affect growth kinetics. (D) ETBF was grown overnight in BHIS supplemented with PBS (-) or 0.5% of various carbohydrates (Glu = glucose, A = arabinose, F = fructose, Gal = galactose, R = rhamnose, L = lactose, S = sorbitol, M = manitol) and cleaved active BFT (BFT*) was probed by western blot from culture supernatants.

Figure 3.

Complex protein-level regulation of BFT by environmental cues. (A) Bacteria from stationary phase cultures of ETBF strain ATCC4 3858 were sedimented, resuspended in spent media from normal growth conditions (UT) or fresh BHIS at pH 5.6 and pH 7.4, incubated for 1 hour at 37°C, and separated into pellet and supernatant fractions. BFT in the supernatant fraction was analyzed by Western blot for full length protoxin (FLBFT, upper band) and cleaved active toxin (BFT*). (B) Heat and oxidative stress upregulate BFT production. ETBF strain ATCC 43858 was grown to late-log phase, a growth phase during which BFT expression is not normally detected. Cultures were then exposed to different conditions for 1 hour: normal growth conditions (UT), 42°C, room temperature (RT), or room air (O²). Cell pellet and supernatant fractions were probed for full length protoxin (FLBFT, upper band) and cleaved active toxin (BFT*). The lower band is nonspecific.

Figure 4.

Spatiotemporal regulation of BFT predicts the outcome of virulence and competition. Bacterial competition during host development drives ETBF into colonic mucus where BFT activity manipulates the carbohydrate composition for its advantage by stimulating host immunity (top panel). This interaction becomes homeostatic during mucus layer maturation as Fragipain deactivates in immature acidic mucus most proximate to the epithelium (middle panel). Depletion of fermentable carbohydrates due to depletion of the mucus layer, or insufficient dietary fiber intake, drives ETBF further into the mucosa, where host-microbe interaction is further dysregulated by aerobic upregulation of BFT (bottom). In this case Fpn deactivation is compensated for by the number of organisms, volume of bft transcription, or host proteases.