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, 22 (5), 563-7

Activation of Bacteroides Fragilis Toxin by a Novel Bacterial Protease Contributes to Anaerobic Sepsis in Mice

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Activation of Bacteroides Fragilis Toxin by a Novel Bacterial Protease Contributes to Anaerobic Sepsis in Mice

Vivian M Choi et al. Nat Med.

Abstract

Bacteroides fragilis is the leading cause of anaerobic bacteremia and sepsis. Enterotoxigenic strains that produce B. fragilis toxin (BFT, fragilysin) contribute to colitis and intestinal malignancy, yet are also isolated in bloodstream infection. It is not known whether these strains harbor unique genetic determinants that confer virulence in extra-intestinal disease. We demonstrate that BFT contributes to sepsis in mice, and we identify a B. fragilis protease called fragipain (Fpn) that is required for the endogenous activation of BFT through the removal of its auto-inhibitory prodomain. Structural analysis of Fpn reveals a His-Cys catalytic dyad that is characteristic of C11-family cysteine proteases that are conserved in multiple pathogenic Bacteroides spp. and Clostridium spp. Fpn-deficient, enterotoxigenic B. fragilis has an attenuated ability to induce sepsis in mice; however, Fpn is dispensable in B. fragilis colitis, wherein host proteases mediate BFT activation. Our findings define a role for B. fragilis enterotoxin and its activating protease in the pathogenesis of bloodstream infection, which indicates a greater complexity of cellular targeting and activity of BFT than previously recognized. The expression of fpn by both toxigenic and nontoxigenic strains suggests that this protease may contribute to anaerobic sepsis in ways that extend beyond its role in toxin activation. It could thus potentially serve as a target for disease modification.

Figures

Fig. 1
Fig. 1. BFT contributes to lethal sepsis. (a)
Kaplan-Meier curves demonstrating survival following intravenous infection of 4 week-old female C57Bl/6 mice with 1 × 10 wild-type (WT) ETBF strain 43859 (n = 9), an isogenic Δbft mutant (n = 10), or the Δbft mutant with genomic complementation of bft (cbft, n = 8). Results are representative of three independent experiments. (b) Kaplan-Meier curves of survival following intravenous injection of 4 week-old female C57Bl/6 mice (n = 5 per group) with 2 μg purified, active BFT or an enzymatically inactive variant (iBFT). **, P < 0.01, ****, P < 0.0001, Mantel-Cox log rank test. Results are representative of two independent experiments.
Fig. 2
Fig. 2. BFT is activated by a novel B. fragilis cysteine protease. (a)
Anti-BFT western blot analysis of 8G7 and 57G6 mutants to evaluate active BFT in supernatants (BFT*). (b) Transposon insertion sites mapped to the intergenic region upstream of a clostripain-related protein in strain NCTC 9343. (c) ClustalX alignment of clostripain-related protein sequence from ETBF (ATCC 43858), NTBF strains (NCTC 9343, YCH46, 638R), other Bacteroides spp., and C. histolyticum clostripain noting the catalytic dyad (asterisks) and the C11 cysteine protease H-G-(Xaa)n-A-C motif (blue boxes). (d) Fpn expression in wild-type (WT) ETBF strain 43859, an isogenic fpn deletion mutant (Δfpn), and the Δfpn mutant with genomic complementation of fpn (cfpn). (e) Western blot analysis of full-length BFT (FL-BFT) expression in cell pellets of WT, Δfpn, and cfpn ETBF strains, and NCTC 9343 (NTBF)(left panel), and analysis of culture supernatants for active BFT (BFT*) (right panel). (f) E-cadherin staining in HT-29 cell monolayers treated with supernatants from WT, Δbft, Δfpn, and cfpn cultures. Green, E-cadherin; blue, nuclei (DAPI). Scale bars = 10 μm. (g) Anti-BFT western blot analysis (upper panel) of reaction products generated following incubation of purified, recombinant forms of Fpn and BFT, a BFT cleavage site mutant (BFTR211A) or predicted Fpn active site dyad mutants (FpnH135A and FpnC180A), noting non-specific cleavage fragments (bracket). N-terminal sequencing of BFT cleavage products (encircled) by Edman degradation and documentation of the Fpn cleavage site in BFT and BFTR211A. Western blot analysis of cleaved E-cadherin in culture supernatants following incubation of HT-29 cells with reaction products from above (lower panel, lanes 3-4).
Fig. 3
Fig. 3. Sequence and molecular structure of B. fragilis fragipain (Fpn). (a)
Fpn amino acid sequence where α-helices are numbered and delimited by a black rectangle and β-strands are numbered and delimited by a black arrow. The active site residues H135 and C180 are boxed in red. Residue S44, which makes contact with the imidazole ring of H135 is boxed in purple. The cleavage site (R147-W148) is boxed in green. (b) X-ray crystal structure of Fpn demonstrating α-helices in purple; β-strands in grey. The active site residues H135 and C180 are in red. The water molecule (W) present in the active site is represented as a red sphere. Residues at the cleavage site (R147-W148) are yellow. (c) Surface representation of Fpn molecules (A and B) in adjacent unit cells; R147 of molecule A is bound in the active site of molecule B. (d) Stereoview of the simulated annealing composite omit map (contoured at 2σ) of the R147 region. (e) Magnified view of the Fpn active site. Residues H135 and C180 are shown as sticks; contact between S44 and H135 is shown as a dotted line. R147 from the molecule in the next unit cell is shown in cyan. The water molecule (W) present in the active site is represented as a red sphere.
Fig. 4
Fig. 4. Selective requirement for Fpn in lethal sepsis. (a)
Colonic tissue sections stained with hematoxylin and eosin 7 days following orogastric inoculation of 4 week-old female C57Bl/6 mice with 1 × 10 wild-type (WT), an isogenic ETBF Δfpn mutant, or the Δfpn with genomic complementation of fpn (cfpn). Scale bar = 100 μm. (b) Colonic weight to length ratio 7 days post-inoculation in mice infected with WT (n = 9), Δbft ETBF (n = 10), Δfpn mutant (n = 9), or the cfpn (n = 10) strains as in (a). ****, P < 0.0001, Student's unpaired two-sided t test. Results are representative of three independent experiments. Error bars, mean ± s.e.m. (c) Analysis of active BFT production (BFT*) following incubation of purified full-length BFT (FL BFT) with colonic mucous prepared in PBS following harvest from specific pathogen-free (SPF) or germ-free (GF) female C57Bl/6 mice. A small amount of BFT cleavage is observed upon incubation with PBS alone (lane 1). Results are representative of two independent experiments. (d) Kaplan-Meier curves demonstrating survival following intravenous infection of 4 week-old female C57Bl/6 mice with 1 × 10 wild-type (WT, n = 7), an Δfpn (n = 8), or cfpn (n = 8) ETBF. **, P < 0.01, ****, P < 0.0001, Mantel-Cox log rank test. Results are representative of three independent experiments. (e) VE-cadherin staining in human pulmonary artery endothelial cell monolayers treated with BFT compared to monolayers treated with media alone or iBFT. Green, VE-cadherin; blue, nuclei (DAPI). Scale bars = 10 μm. Results are representative of two independent experiments.

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