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:	Quanxin Gao, Shaokui Yi, Yang Li, Jinping Luo, Qianqian Xing, Xia Yang, Ming Zhao, Minghua Min, Qian Wang, Yabing Wang, Lingbo Ma, Shiming Peng
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	Vibrio anguillarum flagellin flaB TLR5 structure-activity relationship protein-protein interaction Immunologic diseases. Allergy RC581-607
Acceso en línea:	https://doaj.org/article/b4f3e2f1a05a4e02b095020b767efe55
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spelling	oai:doaj.org-article:b4f3e2f1a05a4e02b095020b767efe552021-12-01T13:58:36ZThe Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification1664-322410.3389/fimmu.2021.774233https://doaj.org/article/b4f3e2f1a05a4e02b095020b767efe552021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fimmu.2021.774233/fullhttps://doaj.org/toc/1664-3224Vibrio 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.Quanxin GaoShaokui YiYang LiJinping LuoQianqian XingXia YangMing ZhaoMinghua MinQian WangYabing WangLingbo MaShiming PengFrontiers Media S.A.articleVibrio anguillarumflagellinflaBTLR5structure-activity relationshipprotein-protein interactionImmunologic diseases. AllergyRC581-607ENFrontiers in Immunology, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Vibrio anguillarum flagellin flaB TLR5 structure-activity relationship protein-protein interaction Immunologic diseases. Allergy RC581-607
spellingShingle	Vibrio anguillarum flagellin flaB TLR5 structure-activity relationship protein-protein interaction Immunologic diseases. Allergy RC581-607 Quanxin Gao Shaokui Yi Yang Li Jinping Luo Qianqian Xing Xia Yang Ming Zhao Minghua Min Qian Wang Yabing Wang Lingbo Ma Shiming Peng The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification
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.
format	article
author	Quanxin Gao Shaokui Yi Yang Li Jinping Luo Qianqian Xing Xia Yang Ming Zhao Minghua Min Qian Wang Yabing Wang Lingbo Ma Shiming Peng
author_facet	Quanxin Gao Shaokui Yi Yang Li Jinping Luo Qianqian Xing Xia Yang Ming Zhao Minghua Min Qian Wang Yabing Wang Lingbo Ma Shiming Peng
author_sort	Quanxin Gao
title	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_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_sort	role of flagellin b in vibrio anguillarum-induced intestinal immunity and functional domain identification
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/b4f3e2f1a05a4e02b095020b767efe55
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The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification

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