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Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from Clostridium difficile Ribotype 027.
mSphere 2019; 4(5)M

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.

Authors+Show Affiliations

Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, USA.Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, USA.Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, USA.Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA.Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USA.Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, USA zchen4@tamu.edu.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

31578248

Citation

Peng, Zeyu, et al. "Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB From Clostridium Difficile Ribotype 027." MSphere, vol. 4, no. 5, 2019.
Peng Z, Simeon R, Mitchell SB, et al. Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from Clostridium difficile Ribotype 027. mSphere. 2019;4(5).
Peng, Z., Simeon, R., Mitchell, S. B., Zhang, J., Feng, H., & Chen, Z. (2019). Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from Clostridium difficile Ribotype 027. MSphere, 4(5), doi:10.1128/mSphere.00596-19.
Peng Z, et al. Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB From Clostridium Difficile Ribotype 027. mSphere. 2019 10 2;4(5) PubMed PMID: 31578248.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from Clostridium difficile Ribotype 027. AU - Peng,Zeyu, AU - Simeon,Rudo, AU - Mitchell,Samuel B, AU - Zhang,Junjie, AU - Feng,Hanping, AU - Chen,Zhilei, Y1 - 2019/10/02/ PY - 2019/10/4/entrez PY - 2019/10/4/pubmed PY - 2019/10/4/medline KW - antibody KW - enterotoxins KW - hypervirulent KW - infection KW - protein KW - therapeutic KW - toxin JF - mSphere JO - mSphere VL - 4 IS - 5 N2 - 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. SN - 2379-5042 UR - https://www.unboundmedicine.com/medline/citation/31578248/Designed_Ankyrin_Repeat_Protein_(DARPin)_Neutralizers_of_TcdB_from_Clostridium_difficile_Ribotype_027 L2 - https://doi.org/10.1128/mSphere.00596-19 DB - PRIME DP - Unbound Medicine ER -