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Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB From Clostridium Difficile Ribotype 027

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Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB From Clostridium Difficile Ribotype 027

Zeyu Peng et al. mSphere.

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.

Keywords: antibody; enterotoxins; hypervirulent; infection; protein; therapeutic; toxin.

Figures

FIG 1
FIG 1
(A) Schematics of the different DARPins. The identity of the randomized residues in each repeat module is indicated in the legend on the right. The different colors represent the different positions on the ankyrin repeat module and are pictorially represented in panel B. The number of clones with the same AR configuration (with or without additional framework mutations) is shown in parentheses. The asterisk (*) indicates a DARPin that contains one framework mutation. (B) A crystal structure illustrating the structure of DARPin. Residues colored in red, green, and blue are randomized in the library.
FIG 2
FIG 2
(A and B) DARPins strongly exhibited the ability to neutralize (A) and bind (B) the different TcdB toxins. conc., concentration. (C) TcdB neutralization potency of different DARPins and bezlotoxumab. For neutralization assays, serially diluted immobilized-metal affinity chromatography (IMAC)-purified DARPins were mixed with the appropriate toxin and then added to Vero cells seeded the night before in 96-well plates. The cell viability was quantified by the CellTiterGlo assay 72 h later and normalized to naive Vero cells. For ELISAs, the MaxiSorp plates were coated with the appropriate toxin followed by treatment with serially diluted DARPins. The amounts of plate-bound DARPins (containing Myc tags) were quantified using an anti-c-Myc antibody. Data in panel A represent averages of results from at least 2 independent experiments. Data presented in panel B are representative of results from two independent experiments performed in duplicate.
FIG 3
FIG 3
Anti-TcdBUK1 DARPins block the interaction between TcdB and its receptor CSPG4. ***, P < 0.001 (t test). The wells of an ELISA plate were coated with TcdBUK1 followed by treatment with a 1 nM concentration of a CSPG4 extracellular domain-GFP fusion protein alone or in a mixture with a 250 nM concentration of the indicated DARPins. The amounts of plate-bound CSPG4 were detected using an anti-GFP antibody. The data are representative of results from two independent experiments and of averages of results from quadruplicate samples.
FIG 4
FIG 4
DARPin dimer U3D16 showed enhanced neutralization ability against TcdBVPI (A) and TcdBM68 (B). Serially diluted DARPins were mixed with the appropriate toxins and then added to Vero cells that had been seeded the night before. Cell viability was quantified by the CellTiterGlo assay 72 h later and normalized to naive Vero cells. The error bars represent mean deviations of results from two independent experiments. (C) U3 lacks the ability to bind to TcdBUK1 as determined by ELISA. The ELISA plates were coated with the appropriate toxin and then blocked with BSA prior to the addition of serially diluted DARPin U3. The amounts of plate-bound DARPin were quantified using an anti-c-Myc antibody. The data are representative of results from two independent experiments performed in duplicate. OD450, optical density at 450 nm.
FIG 5
FIG 5
TcdBUK1 lacks significant ability to interact with FZD2. The wells of an ELISA plate were coated with TcdBVPI or TcdBUK1 followed by treatment with CSPG4-EC-GFP (1 nM) (A), FZD2-Fc (4 nM) (B), or PVRL3 (100 nM) (C). The amount of plate-bound CSPG4, FZD2, or PVRL3 was detected using each of the respective antibodies. Results are representative of at least two independent experiments. Error bars represent the mean deviations of results from duplicate samples.
FIG 6
FIG 6
Vero and Caco-2 cells exhibit different levels of sensitivity to TcdBUK1 and TcdBVPI. Vero cells (A) or Caco-2 cells (B) were incubated with serial dilutions of TcdBUK1 or TcdBVPI. The cell viability was quantified using CellTiter-Glo reagent 72 h later and normalized to naive cells. The error bars represent standard deviations of results from two independent experiments performed in triplicate.
FIG 7
FIG 7
Overlay of homology model of FBD from TcdBUK1 with the crystal structure of TcdBVPI (PDB code: 6C0B) in two different views (A, side view; B, front view). The toxin and the cysteine-rich domain 2 (CRD2) from FZD2 are represented in brown and green, respectively. Key positive allosteric modulator (PAM) (silver) binding residues from the toxin (brown) and CRD2 (green) are shown as stick models. The FBD sequence from TcdBM68 is identical to that from TcdBVPI.

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