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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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2019-10-01
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| Series: | mSphere |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mSphere.00596-19 |
| _version_ | 1828953520574824448 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T07:12:54Z |
| format | Article |
| id | doaj.art-144a26aa4c874833b3124d220b7faf2a |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-12-14T07:12:54Z |
| publishDate | 2019-10-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSphere |
| spelling | doaj.art-144a26aa4c874833b3124d220b7faf2a2022-12-21T23:11:45ZengAmerican Society for MicrobiologymSphere2379-50422019-10-014510.1128/mSphere.00596-19Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from <named-content content-type="genus-species">Clostridium difficile</named-content> Ribotype 027Zeyu Peng0Rudo Simeon1Samuel B. Mitchell2Junjie Zhang3Hanping Feng4Zhilei Chen5Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, USADepartment of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, USADepartment of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, USADepartment of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USADepartment of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USADepartment of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, USAABSTRACT 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.https://journals.asm.org/doi/10.1128/mSphere.00596-19toxintherapeuticinfectionproteinantibodyhypervirulent |
| spellingShingle | 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 mSphere toxin therapeutic infection protein antibody hypervirulent |
| title | 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_short | 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 |
| topic | toxin therapeutic infection protein antibody hypervirulent |
| url | https://journals.asm.org/doi/10.1128/mSphere.00596-19 |
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