Abstract
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.
Highlights
Vibrio anguillarum, one of the most common bacteria in the marine environment, is widely distributed in coastal and estuarine seawater [1]
The results showed that expression of IL-8 at 60 min and tumour necrosis factor a (TNF-a) at 30 min was significantly lower in the mutant group than in the wild-type group in in vitro experiments
Our results showed that treatment with flaB induced the expression of Green fluorescent protein (GFP)-extracellular region of the TLR5 protein (erTLR5) protein (Figure 7C), which further demonstrated an interaction between flaB and TLR5
Summary
One of the most common bacteria in the marine environment, is widely distributed in coastal and estuarine seawater [1]. V. anguillarum is a representative opportunistic bacterial pathogen that causes disease in marine fish. It causes vibriosis in both wild and farmed fish and other aquatic animals, resulting in huge losses to aquaculture [2]. V. anguillarum is considered a significant threat to commercial production in marine aquaculture. At least 50 species of fish can be infected with V. anguillarum, including pacific salmon, Atlantic salmon, rainbow trout, turbot, perch, sea carp, striped bass, codfish and Japanese flounder [3, 4]. In view of the wide host range and economic losses caused by this devastating pathogen, elucidating its infection mechanism is vital for the development of control methods
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
