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Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells
Neisseria adhesin A (NadA), one of the surface adhesins of Neisseria meningitides (NM), interacts with several cell types including human brain microvascular endothelial cells (hBMECs) and play important role in the pathogenesis. Receptor binding pockets of NadA are localized on the globular head do...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785856/ https://www.ncbi.nlm.nih.gov/pubmed/33425985 http://dx.doi.org/10.3389/fmolb.2020.573281 |
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author | Kulkarni, Amod Mochnáčová, Evelína Majerova, Petra Čurlík, Ján Bhide, Katarína Mertinková, Patrícia Bhide, Mangesh |
author_facet | Kulkarni, Amod Mochnáčová, Evelína Majerova, Petra Čurlík, Ján Bhide, Katarína Mertinková, Patrícia Bhide, Mangesh |
author_sort | Kulkarni, Amod |
collection | PubMed |
description | Neisseria adhesin A (NadA), one of the surface adhesins of Neisseria meningitides (NM), interacts with several cell types including human brain microvascular endothelial cells (hBMECs) and play important role in the pathogenesis. Receptor binding pockets of NadA are localized on the globular head domain (A(33) to K(69)) and the first coiled-coil domain (L(121) to K(158)). Here, the phage display was used to develop a variable heavy chain domain (VHH) that can block receptor binding sites of recombinant NadA (rec-NadA). A phage library displaying VHH was panned against synthetic peptides (NadA-gd(A33−K69) or NadA-cc(L121−K158)), gene encoding VHH was amplified from bound phages and re-cloned in the expression vector, and the soluble VHHs containing disulfide bonds were overexpressed in the SHuffle E. coli. From the repertoire of 96 clones, two VHHs (VHH(F3)–binding NadA-gd(A33−K69) and VHH(G9)–binding NadA-cc(L121−K158)) were finally selected as they abrogated the interaction between rec-NadA and the cell receptor. Preincubation of NM with VHH(F3) and VHH(G9) significantly reduced the adhesion of NM on hBMECs in situ and hindered the traversal of NM across the in-vitro BBB model. The work presents a phage display pipeline with a single-round of panning to select receptor blocking VHHs. It also demonstrates the production of soluble and functional VHHs, which blocked the interaction between NadA and its receptor, decreased adhesion of NM on hBMECs, and reduced translocation of NM across BBB in-vitro. The selected NadA blocking VHHs could be promising molecules for therapeutic translation. |
format | Online Article Text |
id | pubmed-7785856 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-77858562021-01-07 Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells Kulkarni, Amod Mochnáčová, Evelína Majerova, Petra Čurlík, Ján Bhide, Katarína Mertinková, Patrícia Bhide, Mangesh Front Mol Biosci Molecular Biosciences Neisseria adhesin A (NadA), one of the surface adhesins of Neisseria meningitides (NM), interacts with several cell types including human brain microvascular endothelial cells (hBMECs) and play important role in the pathogenesis. Receptor binding pockets of NadA are localized on the globular head domain (A(33) to K(69)) and the first coiled-coil domain (L(121) to K(158)). Here, the phage display was used to develop a variable heavy chain domain (VHH) that can block receptor binding sites of recombinant NadA (rec-NadA). A phage library displaying VHH was panned against synthetic peptides (NadA-gd(A33−K69) or NadA-cc(L121−K158)), gene encoding VHH was amplified from bound phages and re-cloned in the expression vector, and the soluble VHHs containing disulfide bonds were overexpressed in the SHuffle E. coli. From the repertoire of 96 clones, two VHHs (VHH(F3)–binding NadA-gd(A33−K69) and VHH(G9)–binding NadA-cc(L121−K158)) were finally selected as they abrogated the interaction between rec-NadA and the cell receptor. Preincubation of NM with VHH(F3) and VHH(G9) significantly reduced the adhesion of NM on hBMECs in situ and hindered the traversal of NM across the in-vitro BBB model. The work presents a phage display pipeline with a single-round of panning to select receptor blocking VHHs. It also demonstrates the production of soluble and functional VHHs, which blocked the interaction between NadA and its receptor, decreased adhesion of NM on hBMECs, and reduced translocation of NM across BBB in-vitro. The selected NadA blocking VHHs could be promising molecules for therapeutic translation. Frontiers Media S.A. 2020-12-23 /pmc/articles/PMC7785856/ /pubmed/33425985 http://dx.doi.org/10.3389/fmolb.2020.573281 Text en Copyright © 2020 Kulkarni, Mochnáčová, Majerova, Čurlík, Bhide, Mertinková and Bhide. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Molecular Biosciences Kulkarni, Amod Mochnáčová, Evelína Majerova, Petra Čurlík, Ján Bhide, Katarína Mertinková, Patrícia Bhide, Mangesh Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells |
title | Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells |
title_full | Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells |
title_fullStr | Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells |
title_full_unstemmed | Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells |
title_short | Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells |
title_sort | single domain antibodies targeting receptor binding pockets of nada restrain adhesion of neisseria meningitidis to human brain microvascular endothelial cells |
topic | Molecular Biosciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785856/ https://www.ncbi.nlm.nih.gov/pubmed/33425985 http://dx.doi.org/10.3389/fmolb.2020.573281 |
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