Double-layered protein nanoparticles conjugated with truncated flagellin induce improved mucosal and systemic immune responses in mice.

Influenza viral infection poses a severe risk to global public health. Considering the suboptimal protection provided by current influenza vaccines against circulating influenza A viruses, it is imperative to develop novel vaccine formulations to combat respiratory infections. Here, we report the development of an intranasally-administered, self-adjuvanted double-layered protein nanoparticle consisting of influenza nucleoprotein (NP) cores coated with hemagglutinin (HA) and a truncated form of bacterial flagellin (tFliC). Intranasal vaccination of these nanoparticles notably amplified both antigen-specific humoral and cellular immune responses in the systematic compartments. Elevated antigen-specific IgA and IgG levels in mucosal washes, along with increased lung-resident memory B cell populations, were observed in the respiratory system of the immunized mice. Furthermore, intranasal vaccination of tFliC-adjuvanted nanoparticles enhanced survival rates against homologous and heterologous H3N2 viral challenges. Intriguingly, mucosal slow delivery of the prime dose (by splitting the dose into 5 applications over 8 days) significantly enhanced germinal center reactions and effector T-cell populations in lung draining lymph nodes, therefore promoting the protective efficacy against heterologous influenza viral challenges compared to single-prime immunization. These findings highlight the potential of intranasal immunization with tFliC-adjuvanted protein nanoparticles to bolster mucosal and systemic immune responses, with a slow-delivery strategy offering a promising approach for combating influenza epidemics.

Harnessing the acceptor substrate promiscuity of Clostridium botulinum Maf glycosyltransferase to glyco-engineer mini-flagellin protein chimeras.

Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5'-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO).

Identification and Functional Characterization of a Surfactant-like Protein Region in Flagellin FliC for Stabilizing Selenium Nanoparticles and Enhancing Bioavailability.

Biogenic selenium nanoparticles (SeNPs) are the most favorable Se form for nutritional supplementation due to their high stability, low toxicity, and high activity. However, the interaction between the surface-binding proteins and their stable biogenic SeNPs, as well as their impact on the stability and bioavailability of SeNPs, remains to be understood. In vitro stabilization experiments revealed an amino acid segment (F(235-386)) in Rahnella aquatilis' flagellin FliC, with surfactant-like properties, stabilizing SeNPs under harsh conditions. FliC and F(235-386) were employed as stabilizers to synthesize SeNPs (FliC@SeNPs and F(235-386)@SeNPs), and surface chemistry analysis revealed coordination reactions between the proteins and Se atoms on the surface of SeNPs. Both FliC and F(235-386) enhanced SeNPs uptake in wheat seedlings but reduced it in bacteria and yeast. This study highlights FliC's core function in stabilizing SeNPs and enhancing their bioavailability, paving the way for agricultural and nutritional applications.

Structural and biochemical basis of FLS2-mediated signal activation and transduction in rice.

The receptor-like kinase FLAGELLIN-SENSITIVE 2 (FLS2) functions as a bacterial flagellin receptor localized on the cell membrane of plants. In Arabidopsis, the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) cooperates with FLS2 to detect the flagellin epitope flg22, resulting in formation of a signaling complex that triggers plant defense responses. However, the co-receptor responsible for recognizing and signaling the flg22 epitope in rice remains to be determined, and the precise structural mechanism underlying FLS2-mediated signal activation and transduction has not been clarified. This study presents the structural characterization of a kinase-dead mutant of the intracellular kinase domain of OsFLS2 (OsFLS2-KDD1013A) in complex with ATP or ADP, resolved at resolutions of 1.98 Å and 2.09 Å, respectively. Structural analysis revealed that OsFLS2 can adopt an active conformation in the absence of phosphorylation, although it exhibits only weak basal catalytic activity for autophosphorylation. Subsequent investigations demonstrated that OsSERK2 effectively phosphorylates OsFLS2, which reciprocally phosphorylates OsSERK2, leading to complete activation of OsSERK2 and rapid phosphorylation of the downstream substrate receptor-like cytoplasmic kinases OsRLCK176 and OsRLCK185. Through mass spectrometry experiments, we successfully identified critical autophosphorylation sites on OsSERK2, as well as sites transphosphorylated by OsFLS2. Furthermore, we demonstrated the interaction between OsSERK2 and OsFLS2, which is enhanced in the presence of flg22. Genetic evidence suggests that OsRLCK176 and OsRLCK185 may function downstream of the OsFLS2-mediated signaling pathway. Our study reveals the molecular mechanism by which OsFLS2 mediates signal transduction pathways in rice and provides a valuable example for understanding RLK-mediated signaling pathways in plants.

The role of flagellin F in Vibrio Parahaemolyticus-induced intestinal immunity and functional domain identification.

The marine pathogen Vibrio parahaemolyticus has caused huge economic losses to aquaculture. Flagellin is a key bacterial virulence factor that induces an inflammatory response via activation of Toll-like receptor 5 (TLR5) signaling. Herein, to explore the inflammatory activity of V. parahaemolyticus flagellins (flaA, flaB, flaC, flaD, flaE, and flaF), we investigated their ability to induce apoptosis in a fish cell line. All six flagellins induced severe apoptosis. Moreover, treatment with V. parahaemolyticus flagellins increased TLR5 and myeloid differentiation factor 88 (MyD88) expression and the production of TNF-α and IL-8 significantly. This indicated that flagellins might induce a TLR5-meditated immune response via an MyD88-dependent pathway. FlaF exhibited the strongest immunostimulatory effect; therefore, the interaction between TLR5 and flaF was screened using the yeast two-hybrid system. A significant interaction between the two proteins was observed, indicating that flaF binds directly to TLR5. Finally, the amino acids that participate in the TLR5-flaF interaction were identified using molecular simulation, which indicated three binding sites. These results deepen our understanding of the immunogenic properties of flagellins from V. parahaemolyticus, which could be used for vaccine development in the future.

Deimmunization of flagellin adjuvant for clinical application.

Flagellin is the cognate ligand for host pattern recognition receptors, toll-like receptor 5 (TLR5) in the cell surface, and NAIP5/NLRC4 inflammasome in the cytosol. TLR5-binding domain is located in D1 domain, where crucial amino acid sequences are conserved among diverse bacteria. The highly conserved C-terminal 35 amino acids of flagellin were proved to be responsible for the inflammasome activation by binding to NAIP5. D2/D3 domains, located in the central region and exposed to the outside surface of flagellar filament, are heterogeneous across bacterial species and highly immunogenic. Taking advantage of TLR5- and NLRC4-stimulating activities, flagellin has been actively developed as a vaccine adjuvant and immunotherapeutic. Because of its immunogenicity, there exist worries concerning diminished efficacy and possible reactogenicity after repeated administration. Deimmunization of flagellin derivatives while preserving the TLR5/NLRC4-mediated immunomodulatory activity should be the most reasonable option for clinical application. This review describes strategies and current achievements in flagellin deimmunization.

Purification and CryoEM Image Analysis of the Bacterial Flagellar Filament.

The bacterial flagellum is a large assembly of about 30 different proteins and is divided into three parts: the filament that acts as a screw propeller, the hook as a universal joint, and the basal body as a rotary motor. In the case of Salmonella, the filament length is 10-15 µm, which is more than ten times longer than the size of the cell. The filament is composed of only one component protein, flagellin, and is made of 11 protofilaments. The filament can form 12 different supercoiled structures as polymorphic forms. Each protofilament can take either the L (left-handed) or R (right-handed) state, and the number ratio of the protofilaments in these two states determines the shape of the supercoil. Some point mutations in flagellin make the filament straight by making all the protofilaments in one of the two states. The straight filaments enable us to use their helical symmetries for structural analysis by electron cryomicroscopy (cryoEM) and single particle image analysis. Here, we describe the methods for the purification of the flagellar filament and cryoEM data collection and image analysis.

In silico peptide-based therapeutics against human colorectal cancer by the activation of TLR5 signaling pathways.

OBJECTIVE: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in both men and women. Toll-like receptor 5 (TLR5), an autoimmune signaling receptor that plays a role in cancer, can be exploited for the suppression of human colon cancer. Salmonella flagellin protein, a novel agonist of TLR5 activating downstream signaling, could be a basis for designing anticancer peptides. METHODS: The three-dimensional crystal structure of TLR5 (PDB ID: 3J0A, Resolution = 26.0 Å) was optimized using the AMBER force field in the YASARA suit. In silico enzymatic digestion tool, PeptideCutter, was used to identify peptides from Salmonella flagellin, an agonist against human TLR5. The 3D structure of the peptides was generated using PEP-FOLD3. These peptides were screened against human TLR5 using shape complementarity principles based on the binding affinity and interactions with the active residue of TLR5 monomer, and the selected peptides were further validated by molecular dynamic (MD) simulation. RESULTS: In this study, we generated 42 peptides from Salmonella flagellin protein by in silico protein digestion. Then, based on a new hidden Markov model sub-optimal conformation sampling approach as well as the size of the fragments, we select 38 effective peptides from these 42 cleavages. These peptides were screened against the monomeric Xray structure of human TLR5 using shape complementarity principles. Based on the binding affinity and interactions with the active residue of TLR5 monomer (residues 294 and 366 of TLR5), nine top-scored peptides were selected for the initial molecular dynamic (MD) simulation. Among these peptides, Clv10, Clv17, and Clv28 showed high stability and less flexibility during MD simulation. A 1 µs MD simulation was performed on TLR5-Clv10, TLR-Clv17, and TLR5-Clv28 complexes to further analyze the stability, conformational changes, and binding mode (Clv10, Clv17, and Clv28). During this MD study, the peptides showed high salt bridges and ionic interactions with residue ASP294 and residue ASP366 throughout the simulation and remained in the concave of the human TLR5 monomer. The RMSD and Rg values showed that the peptide-protein complexes become stable after 200 ns of contraction and extraction. CONCLUSION: These findings can facilitate the rational design of selected peptides as an agonist of TLR5, which have antitumor activity, suppress colorectal cancer tumors, and can be used as promising candidates and novel agonists of TLR5.

Stimulation of naïve B cells with a fusion protein consisting of FlaA and Bet v 1 induces regulatory B cells ex vivo.

BACKGROUND: The experimental fusion protein rFlaA:Betv1 was shown to efficiently suppress allergen-specific sensitization in mice. However, the detailed mechanism of rFlaA:Betv1-mediated immune modulation is not fully understood. In this study, we investigated the effect of rFlaA:Betv1 on naïve murine B cells. METHODS: Immune modulating capacity of rFlaA:Betv1 was screened in IL-10 reporter mice. B cells were isolated from spleens of naïve C57Bl/6, TLR5-/- , or MyD88-/- mice, stimulated with rFlaA:Betv1 and controls, and monitored for the expression of the regulatory B cell markers CD1d, CD24, CD38, and surface IgM by flow cytometry. Secreted cytokines, antibodies, and reactivity of the induced antibodies were investigated by ELISA and intracellular flow cytometry. Suppressive capacity of rFlaA:Betv1-stimulated B cells was tested in mDC:CD4+ T cell:B cell triple cultures. RESULTS: Upon in vivo application of rFlaA:Betv1 into IL-10-GFP reporter mice, CD19+ B cells were shown to produce anti-inflammatory IL-10, suggesting B cells to contribute to the immune-modulatory properties of rFlaA:Betv1. rFlaA:Betv1-induced IL-10 secretion was confirmed in human B cells isolated from buffy coats. In vitro stimulation of naïve murine B cells with rFlaA:Betv1 resulted in an mTOR- and MyD88-dependent production of IL-10 and rFlaA:Betv1 induced Bet v 1-reactive IgG production, which was not observed for IgA. rFlaA:Betv1-stimulated B cells formed a CD19+ CD24+ CD1d+ IgM+ CD38+ Breg subpopulation capable of suppressing Bet v 1-induced TH2 cytokine secretion in vitro. CONCLUSION: rFlaA:Betv1 can act as a thymus-independent B cell antigen, stimulating the mTOR- and MyD88-dependent differentiation of B cells displaying a regulatory phenotype, IL-10 secretion, antigen-binding antibody production, and a suppressive capacity in vitro.

Flagellin outer domain dimerization modulates motility in pathogenic and soil bacteria from viscous environments.

Flagellar filaments function as the propellers of the bacterial flagellum and their supercoiling is key to motility. The outer domains on the surface of the filament are non-critical for motility in many bacteria and their structures and functions are not conserved. Here, we show the atomic cryo-electron microscopy structures for flagellar filaments from enterohemorrhagic Escherichia coli O157:H7, enteropathogenic E. coli O127:H6, Achromobacter, and Sinorhizobium meliloti, where the outer domains dimerize or tetramerize to form either a sheath or a screw-like surface. These dimers are formed by 180° rotations of half of the outer domains. The outer domain sheath (ODS) plays a role in bacterial motility by stabilizing an intermediate waveform and prolonging the tumbling of E. coli cells. Bacteria with these ODS and screw-like flagellar filaments are commonly found in soil and human intestinal environments of relatively high viscosity suggesting a role for the dimerization in these environments.

An archaellum filament composed of two alternating subunits.

Archaea use a molecular machine, called the archaellum, to swim. The archaellum consists of an ATP-powered intracellular motor that drives the rotation of an extracellular filament composed of multiple copies of proteins named archaellins. In many species, several archaellin homologs are encoded in the same operon; however, previous structural studies indicated that archaellum filaments mainly consist of only one protein species. Here, we use electron cryo-microscopy to elucidate the structure of the archaellum from Methanocaldococcus villosus at 3.08 Å resolution. The filament is composed of two alternating archaellins, suggesting that the architecture and assembly of archaella is more complex than previously thought. Moreover, we identify structural elements that may contribute to the filament's flexibility.

The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification.

Vibrio anguillarum, an opportunistic pathogen of aquatic animals, moves using a filament comprised of polymerised flagellin proteins. Flagellins are essential virulence factors for V. anguillarum infection. Herein, we investigated the effects of flagellins (flaA, flaB, flaC, flaD and flaE) on cell apoptosis, TLR5 expression, and production of IL-8 and TNF-α. FlaB exhibited the strongest immunostimulation effects. To explore the functions of flaB in infection, we constructed a flaB deletion mutant using a two-step recombination method, and in vitro experiments showed a significant decrease in the expression of TLR5 and inflammatory cytokines compared with wild-type cells. However in the in vivo study, expression of inflammatory cytokines and intestinal mucosal structure showed no significant differences between groups. Additionally, flaB induced a significant increase in TLR5 expression based on microscopy analysis of fluorescently labelled TLR5, indicating interactions between the two proteins, which was confirmed by native PAGE and yeast two-hybrid assay. Molecular simulation of interactions between flaB and TLR5 was performed to identify the residues involved in binding, revealing two binding sites. Then, based on molecular dynamics simulations, we carried out thirteen site-directed mutations occurring at the amino acid sites of Q57, N83, N87, R91, D94, E122, D152, N312, R313, N320, L97, H316, I324 in binding regions of flaB protein by TLR5, respectively. Surface plasmon resonance (SPR) was employed to compare the affinities of flaB mutants for TLR5, and D152, D94, I324, N87, R313, N320 and H316 were found to mediate interactions between flaB and TLR5. Our comprehensive and systematic analysis of V. anguillarum flagellins establishes the groundwork for future design of flagellin-based vaccines.

Flagellin lysine methyltransferase FliB catalyzes a [4Fe-4S] mediated methyl transfer reaction.

The methyltransferase FliB posttranslationally modifies surface-exposed ɛ-N-lysine residues of flagellin, the protomer of the flagellar filament in Salmonella enterica (S. enterica). Flagellin methylation, reported originally in 1959, was recently shown to enhance host cell adhesion and invasion by increasing the flagellar hydrophobicity. The role of FliB in this process, however, remained enigmatic. In this study, we investigated the properties and mechanisms of FliB from S. enterica in vivo and in vitro. We show that FliB is an S-adenosylmethionine (SAM) dependent methyltransferase, forming a membrane associated oligomer that modifies flagellin in the bacterial cytosol. Using X-band electron paramagnetic resonance (EPR) spectroscopy, zero-field 57Fe Mössbauer spectroscopy, methylation assays and chromatography coupled mass spectrometry (MS) analysis, we further found that FliB contains an oxygen sensitive [4Fe-4S] cluster that is essential for the methyl transfer reaction and might mediate a radical mechanism. Our data indicate that the [4Fe-4S] cluster is coordinated by a cysteine rich motif in FliB that is highly conserved among multiple genera of the Enterobacteriaceae family.

Metabolic Labeling of Legionaminic Acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase.

Campylobacter jejuni is the major human food-borne pathogen. Its bipolar flagella are heavily O-glycosylated with microbial sialic acids and essential for its motility and pathogenicity. However, both the glycosylation of flagella and the exact contribution of legionaminic acid (Leg) to flagellar activity is poorly understood. Herein, we report the development of a metabolic labeling method for Leg glycosylation on bacterial flagella with probes based on azide-modified Leg precursors. The hereby azido-Leg labeled flagellin could be detected by Western blot analysis and imaged on intact bacteria. Using the probes on C. jejuni and its isogenic maf4 mutant we also further substantiated the identification of Maf4 as a putative Leg glycosyltransferase. Further evidence was provided by UPLC-MS detection of labeled CMP-Leg and an in silico model of Maf4. This method and the developed probes will facilitate the study of Leg glycosylation and the functional role of this modification in C. jejuni motility and invasiveness.

Skin vaccination with dissolvable microneedle patches incorporating influenza neuraminidase and flagellin protein nanoparticles induces broad immune protection against multiple influenza viruses.

We generated self-adjuvanted protein nanoparticles of conserved influenza antigens and immunized mice via skin vaccination with dissolvable microneedle patches (MNPs) to increase the strength and breadth of immune responses. We produced M2e nanoparticles via ethanol desolvation, and double-layered NA1/M2e (shell/core), NA1-FliC/M2e, NA2/M2e, and NA2-FliC/M2e protein nanoparticles by chemically crosslinking influenza NA and flagellin (FliC) onto the surfaces of the M2e nanoparticles. The resulting nanoparticles retained FliC TLR5 innate signaling activity and significantly increased antigen-uptake and dendritic cell maturation in vitro. We incorporated the nanoparticles into MNPs for skin vaccination in mice. The nanoparticle MNPs significantly increased M2e and NA-specific antibody levels, the numbers of germinal center B cells, and IL-4 positive splenocytes. Double-layered nanoparticle MNP skin vaccination protected mice against homologous and heterosubtypic influenza viruses. Our results demonstrated that MNP skin vaccination of NA-FliC/M2e nanoparticles could be developed into a standalone or synergistic component of a universal influenza vaccine strategy.

Anesthetics isoflurane and sevoflurane attenuate flagellin-mediated inflammation in the lung.

Isoflurane and sevoflurane are volatile anesthetics (VA) widely used in clinical practice to provide general anesthesia. We and others have previously shown that VAs have immunomodulatory effects and may have a significant impact on the progression of disease states. Flagellin is a component of Gram negative bacteria and plays a significant role in the pathophysiology of bacterial pneumonia through its binding to Toll-like Receptor 5 (TLR5). Our results showed that VAs, not an intravenous anesthetic, significantly attenuated the activation of TLR5 and the release of the neutrophil chemoattractant IL-8 from lung epithelial cells. Furthermore, flagellin-induced lung injury was significantly attenuated by VAs by inhibiting neutrophil migration to the bronchoalveolar space. The lungs of cystic fibrosis (CF) patients are highly colonized by Pseudomonas aeruginosa, which causes inflammation. The retrospective study of oxygenation in patients with CF who had received VA versus intravenous anesthesia suggested that VAs might have the protective effect for gas exchange. To understand the interaction between VAs and TLR5, a docking simulation was performed, which indicated that isoflurane and sevoflurane docked into the binding interphase between TLR5 and flagellin.

Role of DegQ in differential stability of flagellin subunits in Vibrio vulnificus.

Biofilm formation of Vibrio vulnificus is initiated by adherence of flagellated cells to surfaces, and then flagellum-driven motility is not necessary during biofilm maturation. Once matured biofilms are constructed, cells become flagellated and swim to disperse from biofilms. As a consequence, timely regulations of the flagellar components' expression are crucial to complete a biofilm life-cycle. In this study, we demonstrated that flagellins' production is regulated in a biofilm stage-specific manner, via activities of a protease DegQ and a chaperone FlaJ. Among four flagellin subunits for V. vulnificus filament, FlaC had the highest affinities to hook-associated proteins, and is critical for maturating flagellum, showed the least susceptibility to DegQ due to the presence of methionine residues in its DegQ-sensitive domains, ND1 and CD0. Therefore, differential regulation by DegQ and FlaJ controls the cytoplasmic stability of flagellins, which further determines the motility-dependent, stage-specific development of biofilms.

Ralstonia solanacearum type III effector RipV2 encoding a novel E3 ubiquitin ligase (NEL) is required for full virulence by suppressing plant PAMP-triggered immunity.

Ralstonia solanacearum causes bacterial wilt disease in a broad range of plants, primarily through type Ⅲ secreted effectors. However, the R. solanacearum effectors promoting susceptibility in host plants remain limited. In this study, we determined that the R. solanacearum effector RipV2 functions as a novel E3 ubiquitin ligase (NEL). RipV2 was observed to be locali in the plasma membrane after translocatio into plant cells. Transient expression of RipV2 in Nicotiana benthamiana could induce cell death and suppress the flg22-induced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, mediating such effects as attenuation of the expression of several PTI-related genes and ROS bursts. Furthermore, we demonstrated that the conserved catalytic residue is highly important for RipV2. Transient expression of the E3 ubiquitin ligase catalytic mutant RipV2 C403A alleviated the PTI suppression ability and cell death induction, indicating that RipV2 requires its E3 ubiquitin ligase activity for its role in plant-microbe interactions. More importantly, mutation of RipV2 in R. solanacearum reduces the virulence of R. solanacearum on potato. In conclusion, we identified a NEL effector that is required for full virulence of R. solanacearum by suppressing plant PTI.

Ligilactobacillus agilis BKN88 possesses thermo-/acid-stable heteropolymeric flagellar filaments.

Many flagellated bacteria possess multiple flagellins, but the roles and the compositions of each flagellin are diverse and poorly understood. In Ligilactobacillus agilis BKN88, there are two active flagellin gene paralogues but their function and composition in its flagellar filaments have not been described. The aim of this study is to find the function and composition of the flagellins by employing mutant strains each of which expresses a single flagellin or a modified flagellin. Two single flagellin-expressing strains were both flagellated while the number of flagella per cell in the single flagellin-expressing derivatives was lower than that in the wild type. Nonetheless, these derivative strains were apparently equally motile as the wild type. This indicates that either flagellin is sufficient for cell motility. The immunological activity via Toll-like receptor 5 of the single flagellin-expressing strains or purified single flagellins was readily detectable but mostly variably weaker than that of the wild type. The flagellar filaments of wild type L. agilis BKN88 were more acid-/thermo-stable than those of single flagellin-expressing derivatives. Using a combination of immunoprecipitation and flagellin-specific staining, wild type BKN88 appeared to possess heteropolymeric flagellar filaments consisting of both flagellins and each flagellin appeared to be equally distributed throughout the filaments. The results of this study suggest that the two flagellins together form a more robust filament than either alone and are thus functionally complementary.

A structure-function study of C-terminal residues predicted to line the export channel in Salmonella Flagellin.

BACKGROUND: Structural studies of a Salmonella Typhimurium flagellin protein indicated that four polar or charged C-terminal amino acid residues line the inner channel of the flagellum. The hydrophilic character of these putative channel-lining residues was predicted to be essential to facilitate the transport of unfolded flagellin monomers during flagellar assembly. The structure-function relationship of these putative channel-lining residues was investigated by site-directed mutagenesis to examine effects of side chain polarity and size on flagella assembly and function. METHODS: Channel-lining residue variants were generated using site-directed mutagenesis to substitute alanine and other residues to examine the effects of altered side-chain polarity on export and assembly. The export, in vivo motility function, and flagellar structure of variants was characterized by agar motility, video microscopy, immunofluorescence, and SDS-PAGE. RESULTS: Alanine substitution yielded decreased motility and flagellar assembly for three of the four residues. However, alanine substitution of residue Arg 494 did not alter export, although substitution with negatively charged glutamate decreased motility and flagellar filament length. Furthermore, many of the C-terminal mutations affected flagellar filament morphology and stability, often resulting in more tightly coiled and/or more brittle flagella than the wild type. CONCLUSIONS: The four channel-lining C-terminal residues may facilitate monomer protein transport but also have structural roles in determining the stability and morphology of the flagellum. GENERAL SIGNIFICANCE: These results provide further insight into the complex process of bacterial flagellin export and flagellar assembly and provide evidence of previously unknown structural functions for the four putative channel-lining residues.