Abstract
Clostridium difficile infection (CDI) is a leading cause of hospital-acquired diarrhea. In recent decades, the emergence of the "hypervirulent" BI/NAP1/027 strains of C. difficile significantly increased the morbidity and mortality of CDI. The pathogenesis of CDI is primarily mediated by the action of two toxins, TcdA and TcdB, with TcdB being the major virulent factor in humans. In this report, we describe the engineering of a panel of designed ankyrin repeat proteins (DARPins) that potently neutralize TcdB from the BI/NAP1/027 strains (e.g., TcdBUK1). The most effective DARPin, D16, inhibits TcdBUK1 with a 50% effective concentration (EC50) of 0.5 nM, which is >66-fold lower than that of the FDA-approved anti-TcdB antibody bezlotoxumab (EC50, ∼33 nM). Competitive enzyme-linked immunosorbent assays (ELISAs) showed that D16 blocks interactions between TcdB and its receptor, chondroitin sulfate proteoglycan 4 (CSPG4). The dimeric DARPin U3D16, which pairs D16 with DARPin U3, a disrupter of the interaction of TcdB with Frizzled 1/2/7 receptor, exhibits 10-fold-to-20-fold-enhanced neutralization potency against TcdB from C. difficile strains VPI 10463 (laboratory strain) and M68 (CF/NAP9/017) but identical activity against TcdBUK1 relative to D16. Subsequent ELISAs revealed that TcdBUK1 did not significantly interact with Frizzled 1/2/7. Computation modeling revealed 4 key differences at the Frizzled 1/2/7 binding interface which are likely responsible for the significantly reduced binding affinity.IMPORTANCE We report the engineering and characterization of designed ankyrin proteins as potent neutralizers of TcdB toxin secreted by a hypervirulent ribotype 027 strain of Clostridium difficile We further show that although TcdB toxins from both ribotype 027 and VPI 10461 interact efficiently with TcdB receptors CSPG4 and Pvrl3, TcdB027 lacks significant ability to bind the only known physiologically relevant TcdB receptor, Frizzled 1/2/7.
Highlights
Clostridium difficile infection (CDI) is a leading cause of hospital-acquired diarrhea
Cryo-electron microscopy (Cryo-EM) structural studies combined with competitive enzyme-linked immunosorbent assays (ELISAs) revealed that 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) [37] and Frizzled receptor 1/2/7 (FZD1/2/7) [38], respectively
designed ankyrin repeat proteins (DARPins) were designed based on repeat modules of natural ankyrin proteins and consist of an N-terminal capping repeat (N-cap), three (N3C) internal ankyrin repeats (ARs), and a C-terminal capping repeat (C-cap) [41]
Summary
Clostridium difficile infection (CDI) is a leading cause of hospital-acquired diarrhea. We describe the engineering of a panel of designed ankyrin repeat proteins (DARPins) that potently neutralize TcdB from the BI/NAP1/027 strains (e.g., TcdBUK1). The dimeric DARPin U3D16, which pairs D16 with DARPin U3, a disrupter of the interaction of TcdB with Frizzled 1/2/7 receptor, exhibits 10-fold-to-20-fold-enhanced neutralization potency against TcdB from C. difficile strains VPI 10463 (laboratory strain) and M68 (CF/NAP9/017) but identical activity against TcdBUK1 relative to D16. IMPORTANCE We report the engineering and characterization of designed ankyrin proteins as potent neutralizers of TcdB toxin secreted by a hypervirulent ribotype 027 strain of Clostridium difficile. Bezlotoxumab exhibits potent neutralization activity against TcdB from a broad range of C. difficile strains, its potency is significantly (ϳ185-fold) weaker against toxin from C. difficile 027 than against that from laboratory strain VPI 10463 [33]. Critical sequence differences at the CSPG4 and FZD1/2/7 interacting regions render antiTcdBVPI DARPins powerless against TcdBUK1
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