Abstract
Flagellin is the major structural protein of the flagella of Gram-negative bacteria. Recent work has demonstrated that flagellin is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms, including both mammals and plants. In several different human epithelial cell lines, this innate immune response involves toll-like receptor 5 (TLR5). The mechanisms by which flagellin activates TLR5 and the importance of this interaction in other model systems of flagellin-induced inflammation remain unknown. In this work, random and site-directed mutagenesis of the inflammatory flagellin from enteroaggregative Escherichia coli identified two regions in the conserved D1 domain that are required for interleukin-8 release and TLR5 activation. In contrast, large regions of the variable domain could be excised without reducing the inflammatory activity. In addition, regions of the protein analogous to epitopes that trigger innate immune responses in plants are not involved in Caco-2 flagellin responses. These results highlight the complexity of the interaction between bacterial flagellin and its eukaryotic recognition partners and provide the basis for further studies to characterize the innate immune response to flagellin.
Highlights
Most Gram-negative bacteria express flagella, surface structures that confer motility
Bacterial flagella from plant pathogens can induce immune responses in plants such as Arabidopsis thaliana and tomato [13,14,15,16]. These findings suggest that recognition of bacterial flagella by the innate immune system is a widespread phenomenon among higher eukaryotes
Using both site-directed and random mutagenesis, we demonstrate that disruption of two specific regions in the constant domains of flagellin eliminates IL-8 induction and toll-like receptor 5 (TLR5) activation
Summary
Two Distinct Regions of Flagellin Are Required for IL-8releasing Activity—Based on prior observations showing that only a subset of E. coli flagellins were inflammatory [19], we initially hypothesized that the IL-8-releasing activity of EAEC 042 flagellin would be dependent on epitopes in the central, variable domain of the molecule To identify these epitopes, six deletion mutants of flagellin were generated. All four of these null mutants were located within a five-residue stretch near the start of the C-terminal constant domain This finding was of particular interest because several natural polymorphisms that alter flagellar filament structure occur in the corresponding region of S. typhimurium FliC [1]. To determine whether the loss of activity could be explained by altered fila-
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