Soluble components of the flagellar export apparatus, FliI, FliJ, and FliH, do not deliver flagellin, the major filament protein, from the cytosol to the export gate.

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook-filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear. We have used continuous ATPase activity measurements and quartz crystal microbalance (QCM) studies to characterize interactions between the soluble export components and flagellin or the FliC:FliS substrate-chaperone complex. As controls, interactions between soluble export component pairs were characterized providing Kd values. FliC or FliC:FliS did not influence the ATPase activity of FliI alone or in complex with FliH and/or FliJ suggesting lack of interaction in solution. Immobilized FliI, FliH, or FliJ did not interact with FliC or FliC:FliS detected by QCM. The lack of interaction in the fluid phase between FliC or FliC:FliS and the soluble export components, in particular with the ATPase FliI, suggests that cells use different mechanisms for the export of late minor substrates, and the major substrate, FliC. It seems that the abundantly produced flagellin does not require the assistance of the soluble export components to efficiently reach the export gate

Bacterial Flagellin-Specific Chaperone FliS Interacts with Anti-Sigma Factor FlgM

Flagella are extracellular organelles that propel bacteria. Each flagellum consists of a basal body, a hook, and a filament. The major protein of the filament is flagellin. Induction of flagellin gene expression coincides with secretion of FlgM. The role of FlgM is to inhibit FliA (σ(28)), a flagellum-specific RNA polymerase responsible for flagellin transcription. To prevent premature polymerization of newly synthesized flagellin molecules, FliS, the flagellin-specific chaperone, binds flagellin and facilitates its export. In this study, the interaction between FlgM and FliS from Salmonella enterica serovar Typhimurium was characterized using gel shift, intrinsic tryptophan fluorescence, circular dichroism, limited proteolysis, and cross-linking. We have demonstrated that (i) FliS and FlgM interact specifically, forming a 1:1 complex, (ii) the FliS binding site on FlgM is proximal to or even overlaps the binding site for FliA, and (iii) FliA competes with FliS for FlgM binding

Hybrid flagellin as a T cell independent vaccine scaffold

BACKGROUND: To extend the potency of vaccines against infectious diseases, vaccines should be able to exploit multiple arms of the immune system. One component of the immune system that is under-used in vaccine design is the subset of B cells known to be capable of responding to repetitive antigenic epitopes and differentiate into plasma cells even in the absence of T cell help (T-independent, TI). RESULTS: To target vaccine responses from T-independent B cells, we reengineered a bacterial Flagellin (FliC) by replacing its exposed D3 domain with a viral envelope protein from Dengue virus (DENV2). The resulting hybrid FliC protein (hFliC) was able to form stable filaments decorated with conformationally intact DENV2 envelope domains. These filaments were not only capable of inducing a T cell-dependent (TD) humoral antibody response, but also significant IgM and IgG3 antibody response in a helper T cell repertoire-restricted transgenic mouse model. CONCLUSIONS: Our results provide proof-of-principle demonstration that a reengineered hybrid FliC could be used as a platform for polymeric subunit vaccines, enhancing T cell-dependent and possibly inducing T-independent antibody responses from B-1 B cells as well. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12896-015-0194-0) contains supplementary material, which is available to authorized users