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 

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

JOURNAL=Frontiers in Immunology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2021.774233

DOI=10.3389/fimmu.2021.774233

ISSN=1664-3224

ABSTRACT=<p><italic>Vibrio anguillarum</italic>, an opportunistic pathogen of aquatic animals, moves using a filament comprised of polymerised flagellin proteins. Flagellins are essential virulence factors for <italic>V. anguillarum</italic> infection. Herein, we investigated the effects of flagellins (<italic>flaA</italic>, <italic>flaB</italic>, <italic>flaC</italic>, <italic>flaD</italic> and <italic>flaE</italic>) on cell apoptosis, TLR5 expression, and production of IL-8 and TNF-α. <italic>FlaB</italic> exhibited the strongest immunostimulation effects. To explore the functions of <italic>flaB</italic> in infection, we constructed a <italic>flaB</italic> deletion mutant using a two-step recombination method, and <italic>in vitro</italic> experiments showed a significant decrease in the expression of TLR5 and inflammatory cytokines compared with wild-type cells. However in the <italic>in vivo</italic> study, expression of inflammatory cytokines and intestinal mucosal structure showed no significant differences between groups. Additionally, <italic>flaB</italic> 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 <italic>flaB</italic> 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 <italic>flaB</italic> protein by TLR5, respectively. Surface plasmon resonance (SPR) was employed to compare the affinities of <italic>flaB</italic> mutants for TLR5, and D152, D94, I324, N87, R313, N320 and H316 were found to mediate interactions between <italic>flaB</italic> and TLR5. Our comprehensive and systematic analysis of <italic>V. anguillarum</italic> flagellins establishes the groundwork for future design of flagellin-based vaccines.</p>