An overview of the structure and function of the flagellar hook FlgE protein.

The flagellum is an important organelle for the survival of bacteria and consists of a basal body, hook, and filament. The FlgE protein is the subunit of the hook that connects the basal body and the filament and determines the motility of bacteria. Also, flgE gene plays an essential role in flagellar biosynthesis, swimming ability and biofilm formation. Although the intact flagella and the major component filament have been extensively studied, so far, little is known about the comprehensive understanding of flagellar hook and FlgE. Here in this review, we summarize the structures of flagellar hook and its subunit FlgE in various species and physiological functions of FlgE, including the hook assembly, the structural characteristics of flagellar hook, the mechanical properties of hook, and the similarities and differences between FlgE (hook) and FlgG (distal rod), with special attention on the interaction of FlgE with other molecules, the antigenicity and pro-inflammatory effect of FlgE, and cross-linking of FlgE in spirochetes. We hope our summary of this review could provide a better understanding of the FlgE protein and provide some useful information for developing new effective antibacterial drugs in the future work.

Neutrophils and IL17A mediate flagellar hook protein FlgE-induced mouse acute lung inflammation.

Bacterial flagellar hook and recombinant flagellar hook protein E (FlgE) were reportedly immunostimulatory in mammalian cells or tissues. Current study focused on the mechanisms underlying FlgE stimulation. In an acute lung injury model induced by intranasal FlgE challenge, neutrophils were the predominant infiltrates in lungs, and depletion of neutrophils with anti-Ly6G antibody attenuated FlgE-induced lung damage. However, the FlgE-induced neutrophils recruitment, neutrophils reactive oxygen species (ROS) generation, and neutrophil extracellular traps (NETs) formation were significantly impaired in Il17a-/- mice compared with those in wild-type (WT) mice. In FlgE-treated lung organoids and isolated neutrophils, the phosphorylation levels of signal transfer and activator of transcription protein 3 (STAT3), which was involved in neutrophils functions, were upregulated, but this upregulation was partly impaired upon IL17A deficiency or by IL6 neutralisation. When neutrophils isolated from WT mice were treated with FlgE, the expression of IL17A/IL17RC was increased, but the activation was blocked by STAT3 inhibitor. The NETs formation in FlgE-treated neutrophils was not affected by the ROS inhibitor or recombinant IL17A alone but partly impaired in the presence of STAT3 pathway inhibition. In conclusion, we propose that the pro-inflammatory activities of FlgE are mediated by activating STAT3 phosphorylation and IL17A/IL17R expression and by promoting a ROS-independent NETs formation.

Rapid detection of flagellated and non-flagellated Salmonella by targeting the common flagellar hook gene flgE.

Salmonella spp. can cause animal and human salmonellosis. In this study, we established a simple method to detect all Salmonella species by amplifying a specific region within the flgE gene encoding the flagellar hook protein. Our preliminary sequence analysis among flagella-associated genes of Salmonella revealed that although Salmonella Gallinarum and Salmonella Pullorum are lacking flagella, they did have flagella-associated genes, including flgE. To investigate in detail, a comparative flgE sequence analysis was conducted using different bacterial strains including flagellated and non-flagellated Salmonella as well as non-Salmonella strains. Two unique regions (481-529 bp and 721-775 bp of the reference sequence) within the flgE open reading frame were found to be highly conserved and specific to all Salmonella species. Next, we designed a pair of PCR primers (flgE-UP and flgE-LO) targeting the above two regions, and performed a flgE-tailored PCR using as template DNA prepared from a total of 76 bacterial strains (31 flagellated Salmonella strains, 26 non-flagellated Salmonella strains, and 19 other non-Salmonella bacteria strains). Results showed that specific positive bands with expected size were obtained from all Salmonella (including flagellated and non-flagellated Salmonella) strains, while no specific product was generated from non-Salmonella bacterial strains. PCR products from the positive bands were confirmed by DNA sequencing. The minimum detection amount for genomic DNA and bacteria cells reached 18.3 pg/µL and 100 colony-forming unit (CFU) per PCR reaction, respectively. Using the flgE-PCR method to detect Salmonella in artificially contaminated milk samples, as low as 1 CFU/mL Salmonella was detectable after an 8-h pre-culture. Meanwhile, the flgE-tailored PCR method was applied to evaluate 247 clinical samples infected with Salmonella from different chicken breeding farms. The detection results indicated that flgE-PCR could be used to specifically detect Salmonella in concordance with the traditional bacterial culture-based detection method. It is worthwhile noticed that identification results using flgE-tailored PCR should be completed within less than 1 day, expanding the result of much faster than the standard method, which took more than 5 days. Overall, the flgE-tailored PCR method can specifically detect flagellated and non-flagellated Salmonella and can serve as a powerful tool for rapid, simple, and sensitive detection of Salmonella species. KEY POINTS : • Targeting flgE gene for all Salmonella spp. found. • The established PCR assay is used to specifically detect all Salmonella spp. • The PCR method is applied to detect clinical Salmonella spp. samples within less than 1 day.

Flagellar Hook Protein FlgE Induces Microvascular Hyperpermeability via Ectopic ATP Synthase ß on Endothelial Surface.

We previously demonstrated the immunostimulatory efficacy of Pseudomonas aeruginosa flagellar hook protein FlgE on epithelial cells, presumably via ectopic ATP synthases or subunits ATP5B on cell membranes. Here, by using recombinant wild-type FlgE, mutant FlgE (FlgEM; bearing mutations on two postulated critical epitopes B and F), and a FlgE analog in pull-down assay, Western blotting, flow cytometry, and ELISA, actual bindings of FlgE proteins or epitope B/F peptides with ATP5B were all confirmed. Upon treatment with FlgE proteins, human umbilical vein endothelial cells (HUVECs) and SV40-immortalized murine vascular endothelial cells manifested decreased proliferation, migration, tube formation, and surface ATP production and increased apoptosis. FlgE proteins increased the permeability of HUVEC monolayers to soluble large molecules like dextran as well as to neutrophils. Immunofluorescence showed that FlgE induced clustering and conjugation of F-actin in HUVECs. In Balb/c-nude mice bearing transplanted solid tumors, FlgE proteins induced a microvascular hyperpermeability in pinna, lungs, tumor mass, and abdominal cavity. All effects observed in FlgE proteins were partially or completely impaired in FlgEM proteins or blocked by pretreatment with anti-ATP5B antibodies. Upon coculture of bacteria with HUVECs, FlgE was detectable in the membrane and cytosol of HUVECs. It was concluded that FlgE posed a pathogenic ligand of ectopic ATP5B that, upon FlgE-ATP5B coupling on endothelial cells, modulated properties and increased permeability of endothelial layers both in vitro and in vivo. The FlgE-ectopic ATP5B duo might contribute to the pathogenesis of disorders associated with bacterial infection or ectopic ATP5B-positive cells.

Living in a Foster Home: The Single Subpolar Flagellum Fla1 of Rhodobacter sphaeroides.

Rhodobacter sphaeroides is an α-proteobacterium that has the particularity of having two functional flagellar systems used for swimming. Under the growth conditions commonly used in the laboratory, a single subpolar flagellum that traverses the cell membrane, is assembled on the surface. This flagellum has been named Fla1. Phylogenetic analyses have suggested that this flagellar genetic system was acquired from an ancient γ-proteobacterium. It has been shown that this flagellum has components homologous to those present in other γ-proteobacteria such as the H-ring characteristic of the Vibrio species. Other features of this flagellum such as a straight hook, and a prominent HAP region have been studied and the molecular basis underlying these features has been revealed. It has also been shown that FliL, and the protein MotF, mainly found in several species of the family Rhodobacteraceae, contribute to remodel the amphipathic region of MotB, known as the plug, in order to allow flagellar rotation. In the absence of the plug region of MotB, FliL and MotF are dispensable. In this review we have covered the most relevant aspects of the Fla1 flagellum of this remarkable photosynthetic bacterium.

Epitope mapping of Salmonella flagellar hook-associated protein, FlgK, with mass spectrometry-based immuno-capture proteomics using chicken (Gallus gallus domesticus) sera.

Salmonella, a Gram-negative rod, is the leading foodborne pathogen associated with human acute bacterial gastroenteritis worldwide. The Salmonella flagellum is responsible for bacterial movement, colonization and invasion in the host gastrointestinal tract. The flagellum has a complex structure, composed of more than 35 proteins. Among them, we were interested in the flagellar hook-associated protein (FlgK), which is an immunodominant protein in chickens. In this communication, we applied mass spectrometry-based proteomics in conjunction with chicken immunized sera to map the linear immunoepitopes in the FlgK protein, validated the epitopes with peptide ELISA, and determined serum reactivity to the epitopes from commercial chickens. We previously demonstrated the FlgK proteins are highly conserved among Salmonella serovars. The rFlgK protein was produced by the recombinant technique, and was able to induce immune response in chickens. Further, this study identified four peptides (AEG, GAQ, TAD and LEI) in the rFlgK protein that were captured by sera from chickens immunized with the rFlgK protein. These four peptides were also reacted to 64 individual serum samples collected from 44 - 52 weeks old chickens, suggesting that these peptides may represent the shared immuno-epitopes on the FlgK protein. The findings of the specific shared linear immuno-epitopes on the FlgK protein in this study provide a rationale for further evaluation to determine their utility as epitope vaccines covering multiple serotypes for chicken immunization, and subsequently, for providing safer poultry products for human consumption.

From crystal structure to in silico epitope discovery in the Burkholderia pseudomallei flagellar hook-associated protein FlgK.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a potentially fatal infection that is endemic in Southeast Asia and Northern Australia that is poorly controlled by antibiotics. Research efforts to identify antigenic components for a melioidosis vaccine have led to the identification of several proteins, including subunits forming the flagella that mediate bacterial motility, host colonization, and virulence. This study focuses on the B. pseudomallei flagellar hook-associated protein (FlgK(Bp)), and provides the first insights into the 3D structure of FlgK proteins as targets for structure-based antigen engineering. The FlgK(Bp) crystal structure (presented here at 1.8-Å resolution) reveals a multidomain fold, comprising two small ß-domains protruding from a large elongated α-helical bundle core. The evident structural similarity to flagellin, the flagellar filament subunit protein, suggests that, depending on the bacterial species, flagellar hook-associated proteins are likely to show a conserved, elongated α-helical bundle scaffold coupled to a variable number of smaller domains. Furthermore, we present immune serum recognition data confirming, in agreement with previous findings, that recovered melioidosis patients produce elevated levels of antibodies against FlgK(Bp), in comparison with seronegative and seropositive healthy subjects. Moreover, we show that FlgK(Bp) has cytotoxic effects on cultured murine macrophages, suggesting an important role in bacterial pathogenesis. Finally, computational epitope prediction methods applied to the FlgK(Bp) crystal structure, coupled with in vitro mapping, allowed us to predict three antigenic regions that locate to discrete protein domains. Taken together, our results point to FlgK(Bp) as a candidate for the design and production of epitope-containing subunits/domains as potential vaccine components.