Flagellin Molecules Research Articles

FlgM is required to evade NLRC4-mediated host protection against flagellated Salmonella.

Salmonella enterica serovar Typhimurium is a leading cause of gastroenteritis worldwide and a deadly pathogen in children, immunocompromised patients, and the elderly. Salmonella induces innate immune responses through the NLRC4 inflammasome, which has been demonstrated to have distinct roles during systemic and mucosal detections of flagellin and non-flagellin molecules. We hypothesized that NLRC4 recognition of Salmonella flagellin is the dominant protective pathway during infection. To test this hypothesis, we used wild-type, flagellin-deficient, and flagellin-overproducing Salmonella to establish the role of flagellin in mediating NLRC4-dependent host resistance during systemic and mucosal infections in mice. We observed that during the systemic phase of infection, Salmonella efficiently evades NLRC4-mediated innate immunity. During mucosal Salmonella infection, flagellin recognition by the NLRC4 inflammasome pathway is the dominant mediator of protective innate immunity. Deletion of flgM results in constitutive expression of flagellin and severely limits systemic and mucosal Salmonella infections in an NLRC4 inflammasome-dependent manner. These data establish that recognition of Salmonella's flagellin by the NLRC4 inflammasome during mucosal infection is the dominant innate protective pathway for host resistance against the enteric pathogen and that FlgM-mediated evasion of the NLRC4 inflammasome enhances virulence and intestinal tissue destruction.

Live-Cell Imaging of the Assembly and Ejection Processes of the Bacterial Flagella by Fluorescence Microscopy.

Bacterial flagella are molecular machines used for motility and chemotaxis. The flagellum consists of a thin extracellular helical filament as a propeller, a short hook as a universal joint, and a basal body as a rotary motor. The filament is made up of more than 20,000 flagellin molecules and can grow to several micrometers long but only 20 nanometers thick. The regulation of flagellar assembly and ejection is important for bacterial environmental adaptation. However, due to the technical difficulty to observe these nanostructures in live cells, our understanding of the flagellar growth and loss is limited. In the last three decades, the development of fluorescence microscopy and fluorescence labeling of specific cellular structure has made it possible to perform the real-time observation of bacterial flagellar assembly and ejection processes. Furthermore, flagella are not only critical for bacterial motility but also important antigens stimulating host immune responses. The complete understanding of bacterial flagellar production and ejection is valuable for understanding macromolecular self-assembly, cell adaptation, and pathogen-host interactions.

The Homologous Components of Flagellar Type III Protein Apparatus Have Acquired a Novel Function to Control Twitching Motility in a Non-Flagellated Biocontrol Bacterium.

The bacterial flagellum is one of the best-studied surface-attached appendages in bacteria. Flagellar assembly in vivo is promoted by its own protein export apparatus, a type III secretion system (T3SS) in pathogenic bacteria. Lysobacter enzymogenes OH11 is a non-flagellated soil bacterium that utilizes type IV pilus (T4P)-driven twitching motility to prey upon nearby fungi for food. Interestingly, the strain OH11 encodes components homologous to the flagellar type III protein apparatus (FT3SS) on its genome, but it remains unknown whether this FT3SS-like system is functional. Here, we report that, despite the absence of flagella, the FT3SS homologous genes are responsible not only for the export of the heterologous flagellin in strain OH11 but also for twitching motility. Blocking the FT3SS-like system by in-frame deletion mutations in either flhB or fliI abolished the secretion of heterologous flagellin molecules into the culture medium, indicating that the FT3SS is functional in strain OH11. A deletion of flhA, flhB, fliI, or fliR inhibited T4P-driven twitching motility, whereas neither that of fliP nor fliQ did, suggesting that FlhA, FlhB, FliI, and FliR may obtain a novel function to modulate the twitching motility. The flagellar FliI ATPase was required for the secretion of the major pilus subunit, PilA, suggesting that FliI would have evolved to act as a PilB-like pilus ATPase. These observations lead to a plausible hypothesis that the non-flagellated L. enzymogenes OH11 could preserve FT3SS-like genes for acquiring a distinct function to regulate twitching motility associated with its predatory behavior.

Flagellin-expressing virus-like particles exhibit adjuvant effects on promoting IgG isotype-switched long-lasting antibody induction and protection of influenza vaccines in CD4-deficient mice

Incorporation of membrane-anchored flagellin molecules into the surfaces of influenza virus-like particles (VLP) was previously reported to promote T helper (Th) 1-biased IgG antibody production and protective efficacy of co-presented vaccine antigens. Herein, we investigated the potential adjuvant effects and mechanisms of flagellin-expressing VLP (FliC-VLP) as an independent component on influenza vaccination in wild-type and mutant mouse models. FliC-VLP adjuvanted influenza vaccination was highly effective in promoting the induction of Th1-biased IgG isotype switched antibodies, enhanced protection, and long-lasting IgG antibody responses in both wild-type and CD4-knockout mice. In contrast, the adjuvant effects of soluble flagellin were Th2-biased and required CD4 T helper cells. The adjuvant effects of FliC-VLP were less dependent on CD4 T cells and flagellin-mediated innate immune signaling pathways. The results suggest that FliC-VLP might play an effective adjuvant role in an immune competent condition as well as in a defect of CD4 T cells.

Flagellin based biomimetic coatings: From cell-repellent surfaces to highly adhesive coatings

Biomimetic coatings with cell-adhesion-regulating functionalities are intensively researched today. For example, cell-based biosensing for drug development, biomedical implants, and tissue engineering require that the surface adhesion of living cells is well controlled. Recently, we have shown that the bacterial flagellar protein, flagellin, adsorbs through its terminal segments to hydrophobic surfaces, forming an oriented monolayer and exposing its variable D3 domain to the solution. Here, we hypothesized that this nanostructured layer is highly cell-repellent since it mimics the surface of the flagellar filaments. Moreover, we proposed flagellin as a carrier molecule to display the cell-adhesive RGD (Arg-Gly-Asp) peptide sequence and induce cell adhesion on the coated surface. The D3 domain of flagellin was replaced with one or more RGD motifs linked by various oligopeptides modulating flexibility and accessibility of the inserted segment. The obtained flagellin variants were applied to create surface coatings inducing cell adhesion and spreading to different levels, while wild-type flagellin was shown to form a surface layer with strong anti-adhesive properties. As reference surfaces synthetic polymers were applied which have anti-adhesive (PLL-g-PEG poly(l-lysine)-graft-poly(ethylene glycol)) or adhesion inducing properties (RGD-functionalized PLL-g-PEG). Quantitative adhesion data was obtained by employing optical biochips and microscopy. Cell-adhesion-regulating coatings can be simply formed on hydrophobic surfaces by using the developed flagellin-based constructs. The developed novel RGD-displaying flagellin variants can be easily obtained by bacterial production and can serve as alternatives to create cell-adhesion-regulating biomimetic coatings. Statement of SignificanceIn the present work, we show for the first time that-an oriented and dense monolayer of flagellin molecules mimics the surface of the bacterial flagellar filament. Consequently, the fabricated layer is completely cell repellent.-By genetically modifying flagellin, we incorporate cell adhesion regulating functionalities into this anti-adhesive coating.-We can easily tune the adhesion of living cells from completely cell repellent to highly adhesive.

Evaluation of immunogenicity of rSeM and use of PAMPs as possible enhancers of the immune response

The immune system is responsible for the first immune response to infection. This response is based, in part, by Toll-Like Receptors (TLR) that detect pathogens and induce an appropriate immune response. These receptors bind to structures called Pathogen Associated Molecular Patterns (PAMPs), including lipopolysaccharide, flagellin, lipoproteins, nucleic acids, and other molecules. The Equine Strangles is a bacterial disease caused by Streptococcus equi subspecie equi (S.equi). This bacterium synthesizes several virulence factors, among these M protein (SeM) that stands out for its high antifagocit potential, having an important role in the pathogenesis, making it a promising antigen vaccine candidate. The purpose of this study was to evaluate the immunogenicity of rSeM and the use of PAMPs as possible potentiators of the immune response. Forty Balb/c female mice were immunized intramuscularly on day 0 and 21 of the experiment. Blood samples were collected on days 0, 14 and 28, and processed in order to obtain serum. The mice were divided equally into four groups. The animals belong in groups 1-2 were immunized with the recombinant BL21 (DE3) strain of E.coli, which was previously cloned to express the protein of interest recombinant M protein (rSeM). In group 1, the strain of E.coli was inactivated using 0.1% formaldehyde and increased by 10% aluminum hydroxide, and group 2 received the same strain, but was not inactivated and adjuvant was not added in the vaccine composition. Group 3 was immunized only with the recombinant M protein of rSeM Purified associated with 10% aluminum hydroxide. Each vaccine dose contains 20 μg of rSeM. Animals of to group 4 were used as control and were inoculated with only PBS1X. The immune response of the animals was evaluated by indirect ELISA. It is noteworthy that the animals were kept and handled in accordance with the legal requirements provided for in the Brazilian National Law for Protection of Experimental Animals. Vaccines containing bacterins, used in group 1 and 2 showed the highest antibody seroconversions compared to the group immunized only with the rSeM recombinant vaccine. These results suggest that group 1 and 2 showed higher antibodies absorbance than group 3 due to the fact that these two groups were immunized with the recombinant BL21 (DE3) strain of E.coli, which presents the PAMPs. This work was funded by Centro de Desenvolvimento Tecnologico (CDTec) from Universidade Federal de Pelotas (UFPel).

Pseudomonas syringae Evades Host Immunity by Degrading Flagellin Monomers with Alkaline Protease AprA

Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and plants. The opportunistic pathogen Pseudomonas aeruginosa secretes an alkaline protease called AprA that degrades flagellin monomers. Here, we show that AprA is widespread among a wide variety of bacterial species. In addition, we investigated the role of AprA in virulence of the bacterial plant pathogen P. syringae pv. tomato DC3000. The AprA-deficient DC3000 ΔaprA knockout mutant was significantly less virulent on both tomato and Arabidopsis thaliana. Moreover, infiltration of A. thaliana Col-0 leaves with DC3000 ΔaprA evoked a significantly higher level of expression of the defense-related genes FRK1 and PR-1 than did wild-type DC3000. In the flagellin receptor mutant fls2, pathogen virulence and defense-related gene activation did not differ between DC3000 and DC3000 ΔaprA. Together, these results suggest that AprA of DC3000 is important for evasion of recognition by the FLS2 receptor, allowing wild-type DC3000 to be more virulent on its host plant than AprA-deficient DC3000 ΔaprA. To provide further evidence for the role of DC3000 AprA in host immune evasion, we overexpressed the AprA inhibitory peptide AprI of DC3000 in A. thaliana to counteract the immune evasive capacity of DC3000 AprA. Ectopic expression of aprI in A. thaliana resulted in an enhanced level of resistance against wild-type DC3000, while the already elevated level of resistance against DC3000 ΔaprA remained unchanged. Together, these results indicate that evasion of host immunity by the alkaline protease AprA is important for full virulence of strain DC3000 and likely acts by preventing flagellin monomers from being recognized by its cognate immune receptor.

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.

Determination of the physiological 2:2 TLR5:flagellin activation stoichiometry revealed by the activity of a fusion receptor

Toll-like receptor 5 (TLR5) recognizes flagellin of most flagellated bacteria, enabling activation of the MyD88-dependent signaling pathway. The recently published crystal structure of a truncated zebrafish TLR5 ectodomain in complex with an inactive flagellin fragment indicated binding of two flagellin molecules to a TLR5 homodimer, however this complex did not dimerize in solution. In the present study, we aimed to determine the physiological stoichiometry of TLR5:flagellin activation by the use of a chimeric protein composed of an active flagellin fragment linked to the N-terminus of human TLR5 (SF-TLR5). This construct was constitutively active. Inactivation by the R90D mutation within flagellin demonstrated that autoactivation of the chimeric protein depended solely on the specific interaction between TLR5 and flagellin. Addition of wild-type hTLR5 substantially lowered autoactivation of SF-TLR5 in a concentration dependent manner, an effect which was reversible by the addition of exogenous Salmonella typhimurium flagellin, indicating the biological activity of a TLR5:flagellin complex with a 2:2 stoichiometry. These results, in addition to the combinations of inactive P736H mutation within the BB-loop of the TIR domain of TLR5 and SF-TLR5, further confirm the mechanism of TLR5 activation.

Dynamic Conformational Changes of Flagellar Filament Observed by High-Pressure Microscopy

The bacterial flagellar motor is a molecular machine that rotates a flagellum in both directions. CCW rotation allows the left-handed helical filaments to form a bundle that propels the cell smoothly, whereas CW rotation of a filament leads to change the shape of filament in right-handed helix and break the bundle, and inhibits smooth swimming of the cell, called a tumble. The switching in the helical structure is thought to be caused by directional mechanical actions arising from abrupt change of exerted torque by the motor rotation. Here, we show that application of pressure can also change the helical structure of flagellar filaments. The flagellar filaments in E. coli cells were fluorescently labeled, and then the images were acquired by using a high-pressure microscope [1, 2] with some modifications. We measured the diameter and pitch of the individual filaments and then classified them into 11 possible waveforms which are predicted from structural data. At 0.1 MPa (ambient pressure), all flagellar filaments formed left-handed helical structure (normal form). At 40 MPa, we found left-handed forms (normal and coiled forms) and right- (curly I (or II)). At 80 MPa, 80% flagellar filaments took curly I (or II) forms. After the pressure was released, most filaments returned to the initial left-handed structures. The application of pressure is thought to enhance the structural fluctuation and/or association of water molecules with the exposed regions of flagellin molecules, and results in switching the helical from left- to right-handed structure.[1] Nishiyama M. and Y. Sowa. 2012. Microscopic Analysis of Bacterial Motility at High Pressure. Biophys. J. 102:1872-1880.[2] Nishiyama M. and S. Kojima. 2012. Bacterial motility measured by a miniature chamber for high-pressure microscopy. Int. J. Mol. Sci. 13:9225-9239.

A “Mechanistic” Explanation of the Multiple Helical Forms Adopted by Bacterial Flagellar Filaments

The corkscrew-like flagellar filaments emerging from the surface of bacteria such as Salmonella typhimurium propel the cells toward nutrient and away from repellents. This kind of motility depends upon the ability of the flagellar filaments to adopt a range of distinct helical forms. A filament is typically constructed from ~30,000 identical flagellin molecules, which self-assemble into a tubular structure containing 11 near-longitudinal protofilaments. A “mechanical” model, in which the flagellin building block has the capacity to switch between two principal interfacial states, predicts that the filament can assemble into a “canonical” family of 12 distinct helical forms, each having unique curvature and twist: these include two “extreme” straight forms having left- and right-handed twists, respectively, and 10 intermediate helical forms. Measured shapes of the filaments correspond well with predictions of the model. This report is concerned with two unanswered questions. First, what properties of the flagellin determine which of the 12 discrete forms is preferred? Second, how does the interfacial “switch” work, at a molecular level? Our proposed solution of these problems is based mainly on a detailed examination of differences between the available electron cryo-microscopy structures of the straight L and R filaments, respectively.

Toll-like receptor 5 forms asymmetric dimers in the absence of flagellin

The structure of full-length human TLR5 determined by electron microscopy single-particle image reconstruction at 26Å resolution shows that TLR5 forms an asymmetric homodimer via ectodomain interactions. The structure shows that like TLR9, TLR5 dimerizes in the absence of ligand. The asymmetry of the dimer suggests that TLR5 may recognize two flagellin molecules cooperatively to establish an optimal flagellin response threshold. A TLR5 homology model was generated and fitted into the electron microscopy structure. All seven predicted N-linked glycosylation sites are exposed on the molecular surface, away from the dimer interface. Glycosylation at the first five sites was confirmed by tandem mass spectrometry. Two aspartate residues proposed to interact with flagellin (Asp294 and Asp366) are sterically occluded by a glycan at position 342. In contrast, the central region of the ectodomains near the dimer interface is unobstructed by glycans. Ligand binding in this region would be consistent with the ligand binding sites of other TLRs.

Enterotoxigenic Escherichia coli EtpA mediates adhesion between flagella and host cells

Adhesion to epithelial cells1 and flagella-mediated motility are critical virulence traits for many Gram-negative pathogens, including enterotoxigenic Escherichia coli (ETEC)2, a major cause of diarrhoea in travellers and children in developing countries3,4. Many flagellated pathogens export putative adhesins belonging to the two-partner secretion (TPS) family5. However, the actual function of these adhesins remains largely undefined. Here we demonstrate that EtpA, a TPS exoprotein adhesin of enterotoxigenic Escherichia coli6, mimics and interacts with highly conserved regions of flagellin, the major subunit of flagella, and that these interactions are critical for adherence and intestinal colonization. Although conserved regions of flagellin are mostly buried in the flagellar shaft7, our results suggest that they are at least transiently exposed at the tips of flagella where they capture EtpA adhesin molecules for presentation to eukaryotic receptors. Similarity of EtpA to molecules encoded by other motile pathogens suggests a potential common paradigm for bacterial adhesion, while participation of conserved regions of flagellin in adherence has implications for development of vaccines for Gram-negative pathogens.

Alternative flagellar filament types in the haloarchaeon Haloarcula marismortui

Many Archaea use rotation of helical flagellar filaments for swimming motility. We isolated and characterized the flagellar filaments of Haloarcula marismortui, an archaeal species previously considered to be nonmotile. Two Haloarcula marismortui phenotypes were discriminated--their filaments are composed predominantly of either FlaB or FlaA2 flagellin, and the corresponding genes are located on different replicons. FlaB and FlaA2 filaments differ in antigenicity and thermostability. FlaA2 filaments are distinctly thicker (20-22 nm) than FlaB filaments (16-18 nm). The observed filaments are nearly twice as thick as those of other characterized euryarchaeal filaments. The results suggest that the helicity of Haloarcula marismortui filaments is provided by a mechanism different from that in the related haloarchaeon Halobacterium salinarum, where 2 different flagellin molecules present in comparable quantities are required to form a helical filament.

A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin

The molecular basis for Toll-like receptor (TLR) recognition of microbial ligands is unknown. We demonstrate that mouse and human TLR5 discriminate between different flagellins, and we use this difference to map the flagellin recognition site on TLR5 to 228 amino acids of the extracellular domain. Through molecular modeling of the TLR5 ectodomain, we identify two conserved surface-exposed regions. Mutagenesis studies demonstrate that naturally occurring amino acid variation in TLR5 residue 268 is responsible for human and mouse discrimination between flagellin molecules. Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition. These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand. These findings suggest that ligand binding at the β sheets results in TLR activation and provide a new framework for understanding TLR–agonist interactions.

Expressed and cryptic flagellin genes in the H44 and H55 type strains of Escherichia coli

Bacterial H antigens are specified by flagellin molecules, which constitute the flagellar filament. Escherichia coli 781-55 and E2987-73 are the type strains for H44 and H55 antigens, respectively. Unlike E. coli K-12, they possess two flagellin genes, fliC and fllA, on their chromosomes. However, they are monophasic, expressing exclusively the fllA genes, which specify the type antigens. In this study, the flagellin genes were cloned from these strains and their structure and expression were analyzed. It was found that the fliC genes encode apparently intact flagellin subunits but possess inefficient sigma28-dependent promoters, which may result in these genes being silent. The chromosomal locations of the fllA genes are approximately, but not exactly, identical with that of the phase-2 flagellin gene, fljB, of diphasic Salmonella strains. However, unlike the Salmonella fljB gene, the invertible H segment and the fljA gene responsible for the control of flagellar phase variation are both absent from the fllA loci. The fllA genes are highly homologous to the E. coli fliC gene but distantly related to the Salmonella fljB gene. These results suggest a hypothesis that the fllA genes may have emerged by an intra-species lateral transfer of the fliC gene. This hypothesis is further supported by the observation that the fllA genes are flanked by several IS elements and located within cryptic prophage elements.

Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins

Basal resistance in plants is induced by flagellin and several other common bacterial molecules and is implicated in the immunity of plants to most bacteria and other microbes. However, basal resistance can be suppressed by effector proteins that are injected by the type III secretion system (TTSS) of pathogens such as Pseudomonas syringae. This study demonstrates that basal resistance in the leaves of Nicotiana benthamiana is accompanied by reduced vascular flow into minor veins. Reduced vascular flow was assayed by feeding leaves, via freshly excised petioles, with 1% (weight in volume, w/v) neutral red (NR) and then observing differential staining of minor veins or altered levels of extractable dye in excised leaf samples. The reduced vascular staining was localized to tissues expressing basal resistance and was observable when resistance was induced by either the non-pathogen Pseudomonas fluorescens, a TTSS-deficient mutant of P. syringae pv. tabaci, or flg22 (a flagellin-derived peptide elicitor of basal resistance). Nicotiana benthamiana leaf areas expressing basal resistance no longer elicited the hypersensitive response when challenge inoculated with P. syringae pv. tomato DC3000. The reduced vascular staining effect was suppressed by wild-type P. syringae pv. tabaci and P. fluorescens heterologously expressing a P. syringae TTSS and AvrPto1(PtoJL1065). TTSS-proficient P. fluorescens was used to test the ability of several P. syringae pv. tomato DC3000 effectors for their ability to suppress the basal resistance-associated reduced vascular staining effect. AvrE(PtoDC3000), HopM1(PtoDC3000) (formerly known as HopPtoM), HopF2(PtoDC3000) (HopPtoF) and HopG1(PtoDC3000) (HopPtoG) suppressed basal resistance by this test, whereas HopC1(PtoDC3000) (HopPtoC) did not. In summary, basal resistance locally alters vascular function and the vascular dye uptake assay should be a useful tool for characterizing effectors that suppress basal resistance.

Evasion of Toll-like receptor 5 by flagellated bacteria

Toll-like receptor 5 (TLR5) recognizes an evolutionarily conserved site on bacterial flagellin that is required for flagellar filament assembly and motility. The alpha and epsilon Proteobacteria, including the important human pathogens Campylobacter jejuni, Helicobacter pylori, and Bartonella bacilliformis, require flagellar motility to efficiently infect mammalian hosts. In this study, we demonstrate that these bacteria make flagellin molecules that are not recognized by TLR5. We map the site responsible for TLR5 evasion to amino acids 89-96 of the N-terminal D1 domain, which is centrally positioned within the previously defined TLR5 recognition site. Salmonella flagellin is strongly recognized by TLR5, but mutating residues 89-96 to the corresponding H. pylori flaA sequence abolishes TLR5 recognition and also destroys bacterial motility. To preserve bacterial motility, alpha and epsilon Proteobacteria possess compensatory amino acid changes in other regions of the flagellin molecule, and we engineer a mutant form of Salmonella flagellin that evades TLR5 but retains motility. These results suggest that TLR5 evasion is critical for the survival of this subset of bacteria at mucosal sites in animals and raise the intriguing possibility that flagellin receptors provided the selective force to drive the evolution of these unique subclasses of bacterial flagellins.

Flagellin identified as dominant antigen in Crohn's disease

It is now recognized that inflammatory bowel disease (IBD) results from an aberrant immunologic response to intestinal microflora in genetically susceptible individuals. However, the specific bacterial products that stimulate this response are largely unknown. In this study, the investigators used serologic expression cloning (involving the screening of DNA expression libraries in lambda phage with defined antisera) to identify commensal bacterial proteins that could contribute to disease pathogenesis in murine models of IBD. This led to the identification of 55 clones, of which 15 were flagellin-like sequences. The investigators focused on analysis of the immune response against two of the flagellin clones, Cbir1 and Fla-X. Antibody reactivity to these proteins was composed of the IgG2a subclass (suggesting TH1 responses) and correlated with disease activity. In addition, they isolated flagellin-specific T-cell clones and demonstrated that they could induce IBD when adoptively transferred into immunocompromised mice. Finally, the investigators demonstrated high levels of serum IgG against specific flagellins in patients with Crohn's disease but not in patients with ulcerative colitis or in healthy individuals. Because the receptor for flagellin is the Toll-like receptor 5, these results not only provide insights into the antigens involved in disease pathogenesis but also highlight the important link between IBD and innate immunity. As such, the results suggest that specific flagellin molecules might represent novel targets for antigen-based therapies for IBD.

The neuA/flmD gene cluster of Helicobacter pylori is involved in flagellar biosynthesis and flagellin glycosylation.

Helicobacter pylori possesses a gene (HP0326/JHP309) homologous to neuA of other bacteria, encoding a cytidyl monophosphate-N-acetylneuraminic acid synthetase-homologous enzyme in its N-terminal portion. We analysed the function of this gene, which is controlled by a flagellar class 2 sigma(54) promoter, in flagellar biosynthesis. HP0326/JHP309 actually represents a bicistronic operon consisting of a neuA and a flmD-like putative glycosyl transferase gene. An isogenic flmD mutant synthesized basal bodies but no filaments, was non-motile, and expressed severely reduced amounts of a FlaA flagellin of reduced molecular mass. FlaA flagellin was found to be glycosylated in its exported form within the flagellar filament, but not inside the cytoplasm. Glycosylated FlaA was not detectable in the flmD mutant. Together with other genes in the H. pylori genome, a proposed function of the neuA/flmD gene products could be to provide a pathway for glycosylation of flagellin and other extracytoplasmic molecules during type III secretion.