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The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification

Vibrio anguillarum, an opportunistic pathogen of aquatic animals, moves using a filament comprised of polymerised flagellin proteins. Flagellins are essential virulence factors for V. anguillarum infection. Herein, we investigated the effects of flagellins (flaA, flaB, flaC, flaD and flaE) on cell a...

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Autores principales: Gao, Quanxin, Yi, Shaokui, Li, Yang, Luo, Jinping, Xing, Qianqian, Yang, Xia, Zhao, Ming, Min, Minghua, Wang, Qian, Wang, Yabing, Ma, Lingbo, Peng, Shiming
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8667730/
https://www.ncbi.nlm.nih.gov/pubmed/34912344
http://dx.doi.org/10.3389/fimmu.2021.774233
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author Gao, Quanxin
Yi, Shaokui
Li, Yang
Luo, Jinping
Xing, Qianqian
Yang, Xia
Zhao, Ming
Min, Minghua
Wang, Qian
Wang, Yabing
Ma, Lingbo
Peng, Shiming
author_facet Gao, Quanxin
Yi, Shaokui
Li, Yang
Luo, Jinping
Xing, Qianqian
Yang, Xia
Zhao, Ming
Min, Minghua
Wang, Qian
Wang, Yabing
Ma, Lingbo
Peng, Shiming
author_sort Gao, Quanxin
collection PubMed
description Vibrio anguillarum, an opportunistic pathogen of aquatic animals, moves using a filament comprised of polymerised flagellin proteins. Flagellins are essential virulence factors for V. anguillarum infection. Herein, we investigated the effects of flagellins (flaA, flaB, flaC, flaD and flaE) on cell apoptosis, TLR5 expression, and production of IL-8 and TNF-α. FlaB exhibited the strongest immunostimulation effects. To explore the functions of flaB in infection, we constructed a flaB deletion mutant using a two-step recombination method, and in vitro experiments showed a significant decrease in the expression of TLR5 and inflammatory cytokines compared with wild-type cells. However in the in vivo study, expression of inflammatory cytokines and intestinal mucosal structure showed no significant differences between groups. Additionally, flaB induced a significant increase in TLR5 expression based on microscopy analysis of fluorescently labelled TLR5, indicating interactions between the two proteins, which was confirmed by native PAGE and yeast two-hybrid assay. Molecular simulation of interactions between flaB and TLR5 was performed to identify the residues involved in binding, revealing two binding sites. Then, based on molecular dynamics simulations, we carried out thirteen site-directed mutations occurring at the amino acid sites of Q57, N83, N87, R91, D94, E122, D152, N312, R313, N320, L97, H316, I324 in binding regions of flaB protein by TLR5, respectively. Surface plasmon resonance (SPR) was employed to compare the affinities of flaB mutants for TLR5, and D152, D94, I324, N87, R313, N320 and H316 were found to mediate interactions between flaB and TLR5. Our comprehensive and systematic analysis of V. anguillarum flagellins establishes the groundwork for future design of flagellin-based vaccines.
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spelling pubmed-86677302021-12-14 The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification Gao, Quanxin Yi, Shaokui Li, Yang Luo, Jinping Xing, Qianqian Yang, Xia Zhao, Ming Min, Minghua Wang, Qian Wang, Yabing Ma, Lingbo Peng, Shiming Front Immunol Immunology Vibrio anguillarum, an opportunistic pathogen of aquatic animals, moves using a filament comprised of polymerised flagellin proteins. Flagellins are essential virulence factors for V. anguillarum infection. Herein, we investigated the effects of flagellins (flaA, flaB, flaC, flaD and flaE) on cell apoptosis, TLR5 expression, and production of IL-8 and TNF-α. FlaB exhibited the strongest immunostimulation effects. To explore the functions of flaB in infection, we constructed a flaB deletion mutant using a two-step recombination method, and in vitro experiments showed a significant decrease in the expression of TLR5 and inflammatory cytokines compared with wild-type cells. However in the in vivo study, expression of inflammatory cytokines and intestinal mucosal structure showed no significant differences between groups. Additionally, flaB induced a significant increase in TLR5 expression based on microscopy analysis of fluorescently labelled TLR5, indicating interactions between the two proteins, which was confirmed by native PAGE and yeast two-hybrid assay. Molecular simulation of interactions between flaB and TLR5 was performed to identify the residues involved in binding, revealing two binding sites. Then, based on molecular dynamics simulations, we carried out thirteen site-directed mutations occurring at the amino acid sites of Q57, N83, N87, R91, D94, E122, D152, N312, R313, N320, L97, H316, I324 in binding regions of flaB protein by TLR5, respectively. Surface plasmon resonance (SPR) was employed to compare the affinities of flaB mutants for TLR5, and D152, D94, I324, N87, R313, N320 and H316 were found to mediate interactions between flaB and TLR5. Our comprehensive and systematic analysis of V. anguillarum flagellins establishes the groundwork for future design of flagellin-based vaccines. Frontiers Media S.A. 2021-11-29 /pmc/articles/PMC8667730/ /pubmed/34912344 http://dx.doi.org/10.3389/fimmu.2021.774233 Text en Copyright © 2021 Gao, Yi, Li, Luo, Xing, Yang, Zhao, Min, Wang, Wang, Ma and Peng https://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 Immunology
Gao, Quanxin
Yi, Shaokui
Li, Yang
Luo, Jinping
Xing, Qianqian
Yang, Xia
Zhao, Ming
Min, Minghua
Wang, Qian
Wang, Yabing
Ma, Lingbo
Peng, Shiming
The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification
title The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification
title_full The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification
title_fullStr The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification
title_full_unstemmed The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification
title_short The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification
title_sort role of flagellin b in vibrio anguillarum-induced intestinal immunity and functional domain identification
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8667730/
https://www.ncbi.nlm.nih.gov/pubmed/34912344
http://dx.doi.org/10.3389/fimmu.2021.774233
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