Abstract
Flagellin is a major component of the flagellar filament. Flagellin also functions as a specific ligand that stimulates innate immunity through direct interaction with Toll-like receptor 5 (TLR5) in the host. Because flagellin activates the immune response, it has been of interest to develop as a vaccine adjuvant in subunit vaccines or antigen fusion vaccines. Despite the widespread application of flagellin fusion in preventing infectious diseases, flagellin-antigen fusion designs have never been biophysically and structurally characterized. Moreover, flagellin from Salmonella species has been used extensively despite containing hypervariable regions not required for TLR5 that can cause an unexpected immune response. In this study, flagellin from Bacillus cereus (BcFlg) was identified as the smallest flagellin molecule containing only the conserved TLR5-activating D0 and D1 domains. The crystal structure of BcFlg was determined to provide a scheme for fusion designs. Through homology-based modeling and comparative structural analyses, diverse fusion strategies were proposed. Moreover, cellular and biophysical analysis of an array of fusion constructs indicated that insertion fusion at BcFlg residues 178–180 does not interfere with the protein stability or TLR5-stimulating capacity of flagellin, suggesting its usefulness in the development and optimization of flagellin fusion vaccines.
Highlights
Flagella enable bacterial locomotion toward favorable conditions or away from unfavorable environments
Through cellular and biophysical analysis of the proposed fusion constructs, we identified a flagellin fusion protein that retains its structural integrity and the Toll-like receptor 5 (TLR5)-stimulatory effect of flagellin
Flagellins from Salmonella species have been extensively studied for therapeutics and vaccines, the hypervariable D2 and D3 domains of flagellin not involved in TLR5 activation can cause unexpected toxic cellular responses in vivo
Summary
Flagella enable bacterial locomotion toward favorable conditions or away from unfavorable environments. The bacterial flagellum transverses from the cytoplasm to the outside of the bacterium and comprises more than 30 different proteins[1,2] Among these diverse flagellar proteins, flagellin is a major component of the flagellum that forms the flagellar filament. The D0 and D1 domains mediate inter-molecular and inter-domain interactions and form the helical stem of the filament[5]. The D2 and D3 domains are not necessary for TLR5-mediated immune response and could induce an unwanted toxic immune response in the host, including in humans. Such considerations indicate that the D2 and D3 domains of flagellin should be removed from a flagellin-antigen fusion protein vaccine. Biophysical and cellular analyses of the flagellin fusion protein should be conducted to assess both the structural integrity of the fusion protein and the TLR5-stimulatory activity of the flagellin adjuvant
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