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Activation of Bacteroides fragilis toxin by a novel bacterial protease contributes to anaerobic sepsis in mice

Abstract

Bacteroides fragilis is the leading cause of anaerobic bacteremia and sepsis1. Enterotoxigenic strains that produce B. fragilis toxin (BFT, fragilysin) contribute to colitis2 and intestinal malignancy3, yet are also isolated in bloodstream infection4,5. 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.

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Figure 1: BFT contributes to lethal sepsis.
Figure 2: BFT is activated by a novel B. fragilis cysteine protease.
Figure 3: Sequence and molecular structure of B. fragilis fragipain (Fpn).
Figure 4: Selective requirement for Fpn in lethal sepsis.

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Acknowledgements

This work was supported by the Department of Pediatrics at the University of Chicago and with a Pilot and Feasibility Award from the Digestive Diseases Research Core Center at the University of Chicago to J.B.W. (NIDDK P30DK42086); by a Burroughs Wellcome Foundation Investigators in the Pathogenesis of Infectious Disease Fellowship (J.B.W.); and by the US National Institutes of Health Medical Scientist Training Program at the University of Chicago (GM007281 to V.M.C. and A.L.H.). We thank D. Hecht (Loyola University) for his generous guidance on the establishment of a B. fragilis program and for B. fragilis strain 638R/TM4000; D. Missiakas (University of Chicago) for α-RpoA antisera; J. Gordon (Washington University) for the pSAM-Bt and pKNOCK vectors and Escherichia coli S17 strain; and Y. Fu (University of Texas Southwestern Medical Center) for germ-free mice.

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V.M.C. developed the experimental concepts and approach, generated all bacterial strains, performed transposon-mutagenesis screening and studies on colonic epithelial injury and sepsis in vivo and in vitro, and contributed to writing the manuscript. J.H. generated and analyzed the Fpn crystal structure and contributed to writing the manuscript. A.L.H. contributed essential bacterial genetic reagents and expertise. W.P.T. performed studies on the endothelium. J.R.T. performed pathology analysis of colonic tissues. S.C. analyzed the Fpn crystal structure and contributed to writing the manuscript. J.B.W. developed the experimental concepts and approach and contributed to writing the manuscript.

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Correspondence to Juliane Bubeck Wardenburg.

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Choi, V., Herrou, J., Hecht, A. et al. Activation of Bacteroides fragilis toxin by a novel bacterial protease contributes to anaerobic sepsis in mice. Nat Med 22, 563–567 (2016). https://doi.org/10.1038/nm.4077

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