Helicobacter pylori infection can cause gastritis, peptic ulcer and can lead to gastric cancer. Lengthy antibiotic therapy does not protect the host against reinfection. H. pylori evolved to evade the recognition of the immune response by modifying several of its components whose orthologous proteins from other bacteria activate the innate immune response. Flagella are essential for the H. pylori effective colonization of human duodenum and stomach. TLR5, a member of the Toll-like receptor family, recognizes flagellin of most bacteria, such as Escherichia coli, but does not recognize the flagellin FlaA of H. pylori. We restored the ability of FlaA for the recognition by TLR5 by engineering a chimeric flagellin, in which both terminal segments of H. pylori flagellin were replaced by the corresponding segments from TLR5-activating E. coli flagellin. Recombinant chimeric flagellin folded correctly and was able to activate TLR5. Significantly increased serum IgG and IgA antibody responses were determined in mice vaccinated with chimeric flagellin in comparison to mice vaccinated with a control protein (FlaA) or negative control. Antibody titers remained high eve n8 months after the last immunization. Antibodies were able to bind native flagellin from H. pylori lysate. Vaccination with chimeric flagellin provided mice with significant protection against H. pylori. The approach of chimeric flagellin can therefore generate effective immunogens that enable activation of innate and adaptive immune response and can be used to construct efficient vaccines against H. pylori or other flagellated bacteria that evade TLR5 recognition.
COBISS.SI-ID: 5023770
Toll-like receptor 4 (TLR4) is involved in activation of innate immune response in a large number of different diseases. Despite numerous studies, the role of separate domains of TLR4 in the regulation of receptor activation is poorly understood. Replacement of the TLR4 ectodomain with LPS-binding proteins MD-2 or CD14 resulted in a robust ligand-independent constitutive activation, comparable to the maximal stimulation of the receptor with LPS. The same effect was achieved by the replacement of the ectodomain with a monomeric fluorescent protein or a 24 kDa gyrase B fragment. This demonstratesan intrinsic dimerization propensity of the transmembrane and cytoplasmic domains of TLR4 and reveals a previously unknown function of the ectodomain in inhibiting spontaneous receptor dimerization. Constitutive activation was abolished by the replacement of ectodomain by a bulkier protein ovalbumin. N-terminal deletion variants of TLR4 revealed that the smallest segment of the ectodomain that already prevents constitutive activitycomprises only 90 residues (542 to 631) out of the total 608 residues.We conclude that TLR4 represents a receptor with low threshold of activation, which can be rapidly activated by the release of inhibition exerted by its ectodomain. This is important for the sensitivity of TLR4 to activation by different agonists. TLR4 ectodomain has multiple roles in enabling ligand regulated activation, providing proper localization, while serving as an inhibitor to prevent spontaneous, ligand-independent dimerization.
COBISS.SI-ID: 4652570