Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from <named-content content-type="genus-species">Clostridium difficile</named-content> Ribotype 027

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 th...

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Autores principales:	Zeyu Peng, Rudo Simeon, Samuel B. Mitchell, Junjie Zhang, Hanping Feng, Zhilei Chen
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	toxin therapeutic infection protein antibody hypervirulent Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/144a26aa4c874833b3124d220b7faf2a
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spelling	oai:doaj.org-article:144a26aa4c874833b3124d220b7faf2a2021-11-15T15:27:32ZDesigned Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from <named-content content-type="genus-species">Clostridium difficile</named-content> Ribotype 02710.1128/mSphere.00596-192379-5042https://doaj.org/article/144a26aa4c874833b3124d220b7faf2a2019-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00596-19https://doaj.org/toc/2379-5042ABSTRACT 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.Zeyu PengRudo SimeonSamuel B. MitchellJunjie ZhangHanping FengZhilei ChenAmerican Society for MicrobiologyarticletoxintherapeuticinfectionproteinantibodyhypervirulentMicrobiologyQR1-502ENmSphere, Vol 4, Iss 5 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	toxin therapeutic infection protein antibody hypervirulent Microbiology QR1-502
spellingShingle	toxin therapeutic infection protein antibody hypervirulent Microbiology QR1-502 Zeyu Peng Rudo Simeon Samuel B. Mitchell Junjie Zhang Hanping Feng Zhilei Chen Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from <named-content content-type="genus-species">Clostridium difficile</named-content> Ribotype 027
description	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.
format	article
author	Zeyu Peng Rudo Simeon Samuel B. Mitchell Junjie Zhang Hanping Feng Zhilei Chen
author_facet	Zeyu Peng Rudo Simeon Samuel B. Mitchell Junjie Zhang Hanping Feng Zhilei Chen
author_sort	Zeyu Peng
title	Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from <named-content content-type="genus-species">Clostridium difficile</named-content> Ribotype 027
title_short	Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from <named-content content-type="genus-species">Clostridium difficile</named-content> Ribotype 027
title_full	Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from <named-content content-type="genus-species">Clostridium difficile</named-content> Ribotype 027
title_fullStr	Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from <named-content content-type="genus-species">Clostridium difficile</named-content> Ribotype 027
title_full_unstemmed	Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from <named-content content-type="genus-species">Clostridium difficile</named-content> Ribotype 027
title_sort	designed ankyrin repeat protein (darpin) neutralizers of tcdb from <named-content content-type="genus-species">clostridium difficile</named-content> ribotype 027
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/144a26aa4c874833b3124d220b7faf2a
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Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from <named-content content-type="genus-species">Clostridium difficile</named-content> Ribotype 027

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