Flagellin Secretion Research Articles

Bacterial motility depends on a critical flagellum length and energy-optimised assembly.

Our study demonstrates how protein secretion of the bacterial flagellum is finely tuned to optimize filament assembly rate and flagellum function while minimizing energy consumption. By measuring flagellar filament lengths and bacterial swimming after initiation of flag-ellum assembly, we were able to establish the minimal filament length necessary for swimming motility, which we rationalized physically as resulting from an elasto-hydrodynamic instability of the swimming cell. Our bio-physical model of flagellum growth further illustrates how the physiological flagellin secretion rate is optimized to maximize filament elongation while conserving energy. These findings illuminate the evolutionary pressures that have shaped the function of the bacterial flagellum and type-III secretion system, driving improvements in bacterial motility and overall fitness.

Base-edited live microbiome therapeutics with target-aid -live biotherapeutic products (LBPS) with synergistic effects of anti-PD-1 antibodies in a syngeneic model of mice.

e15104 Background: Base editing without DNA cleaving is a technique that introduces point mutations using enzymes that induce base conversions. Generally, only the point mutation occurs, achieving a high level of precision. Base editing can easily be applied to bacteria, for which DNA cleaving is often lethal. With Bio Palette's core technology of base editing, Target-AID, we can quickly achieve the required therapeutic effects by making pinpoint modifications to bacteria. Immune checkpoint inhibitors (ICIs) targeting CTLA-4, PD-1, or PD-L1 have shown outstanding therapeutic outcomes in various cancers. However, the therapeutic efficacy of ICIs-based immunotherapy has been limited. Recent studies have revealed that the human gut microbiome controls the efficacy of ICIs. Bacterial flagellin derived from Enterococcus spp. has been suggested to enhance immune responses to cancer through activation of TLR5 signaling (1). As a regulatory mechanism of flagellin secretion in Enterococcus spp, FliD, FlgK, FlgN, or FlgM are known as negative regulators. Thus, if the flagellar expression can be enhanced by making the negative regulator functionally deficient, it can be expected to increase the efficacy and response rate of ICIs when combination with a base-edited strain. Methods: As human TLR5 luciferase reporter assay, HEK293T cells were transfected with human TLR5-expression vector. BALB/cAnN mice (6–8 weeks’ old) were injected subcutaneously with 3 x 106 cells for murine colon cancer cell line CT-26. Six days post inoculation, the mice were randomized based on tumor volume into treatment and vehicle control groups (n = 10 per each group). Anti-mouse PD-1 antibody was dosed 5 mg/kg, q3d, iv, for up to 3 weeks either alone or in combination with each of various strains (1 x 109 CFU/mouse, po). Body weight and tumor size were routinely measured until endpoint. Results: To achieve the increased extracellular secretion of Flagellin, we created fliD dysfunctional strain because deletion of the flagellar cap component FliD was expected to promote extracellular secretion of Flagellin monomers. Comparing the wild-type strains we used, E. casseliflavus activated TLR5 compared to E. gallinarum. E. casseliflavus with a stop codon introduced into the fliD gene, BP5040 strain, was found to increase TLR5 activation by approximately 4.9-fold when compared to wild type strain. In vivo experiments indicated that antitumor effect was observed with BP5040 alone. Strikingly, a remarkable antitumor effect was also demonstrated when BP5040 was combined with an anti-PD-1 antibody. Conclusions: To achieve the desired therapeutic effect, we performed base editing without inserting foreign genes. As a result, BP5040 strain is found to be a potential candidate for the treatment of immuno-oncology, which was created using our LBP platform technology.

TonB systems are required for Aeromonas hydrophila motility by controlling the secretion of flagellin

The TonB system is required for the active transport of iron compounds across the outer membrane in Gram-negative bacteria. Our previous data indicated that three TonB systems act coordinately to contribute to the motility of Aeromonas hydrophila NJ-35. In this study, we found that flagellum biogenesis was defective in the ΔtonB123 mutant. Subcellular localization indicated that the flagellin subunits FlaA and FlaB were trapped in the cytoplasm of ΔtonB123 mutant with reduced molecular mass. Overexpression of FlaA or FlaB in the ΔtonB123 mutant was unable to restore the secretion of flagellin subunits. Further investigation demonstrated that flagellins in the ΔtonB123 mutant showed a weak affinity for the flagellin-specific chaperone FliS, which is necessary for the export of flagellins. Deglycosylation analysis indicated that flagellins in the cytoplasm of the ΔtonB123 mutant were almost nonglycosylated. Our data suggested that disruption of tonB123 impairs the formation of flagella by inhibiting flagellin glycosylation and decreasing the binding affinity of flagellin for the chaperone FliS. Taken together, our findings indicate a new role of the TonB system in flagellar biogenesis in A. hydrophila.

Flagellar biosynthesis exerts temporal regulation of secretion of specific Campylobacter jejuni colonization and virulence determinants

The Campylobacter jejuni flagellum exports both proteins that form the flagellar organelle for swimming motility and colonization and virulence factors that promote commensal colonization of the avian intestinal tract or invasion of human intestinal cells respectively. We explored how the C. jejuni flagellum is a versatile secretory organelle by examining molecular determinants that allow colonization and virulence factors to exploit the flagellum for their own secretion. Flagellar biogenesis was observed to exert temporal control of secretion of these proteins, indicating that a bolus of secretion of colonization and virulence factors occurs during hook biogenesis with filament polymerization itself reducing secretion of these factors. Furthermore, we found that intramolecular and intermolecular requirements for flagellar-dependent secretion of these proteins were most reminiscent to those for flagellin secretion. Importantly, we discovered that secretion of one colonization and virulence factor, CiaI, was not required for invasion of human colonic cells, which counters previous hypotheses for how this protein functions during invasion. Instead, secretion of CiaI was essential for C. jejuni to facilitate commensal colonization of the natural avian host. Our work provides insight into the versatility of the bacterial flagellum as a secretory machine that can export proteins promoting diverse biological processes.

Consequences of flagellin export through the type III secretion system ofPseudomonas syringaereveal a major difference in the innate immune systems of mammals and the model plantNicotiana benthamiana

Bacterial flagellin is perceived as a microbe (or pathogen)-associated molecular pattern (MAMP or PAMP) by the extracellular pattern recognition receptors, FLS2 and TLR5, of plants and mammals respectively. Flagellin accidently translocated into mammalian cells by pathogen type III secretion systems (T3SSs) is recognized by nucleotide-binding leucine-rich repeat receptor NLRC4 as a pattern of pathogenesis and induces a death-associated immune response. The non-pathogen Pseudomonas fluorescens Pf0-1, expressing a Pseudomonas syringae T3SS, and the plant pathogen P. syringae pv. tomato DC3000 were used to seek evidence of an analogous cytoplasmic recognition system for flagellin in the model plant Nicotiana benthamiana. Flagellin (FliC) was secreted in culture and translocated into plant cells by the T3SS expressed in Pf0-1 and DC3000 and in their ΔflgGHI flagellar pathway mutants. ΔfliC and ΔflgGHI mutants of Pf0-1 and DC3000 were strongly reduced in elicitation of reactive oxygen species production and in immunity induction as indicated by the ability of challenge bacteria inoculated 6 h later to translocate a type III effector-reporter and to elicit effector-triggered cell death. Agrobacterium-mediated transient expression in N. benthamiana of FliC with or without a eukaryotic export signal peptide, coupled with virus-induced gene silencing of FLS2, revealed no immune response that was not FLS2 dependent. Transiently expressed FliC from DC3000 and Pectobacterium carotovorum did notinduce cell death in N. benthamiana, tobacco or tomato leaves. Flagellin is the major Pseudomonas MAMP perceived by N. benthamiana, and although flagellin secretion through the plant cell wall by the T3SS may partially contribute to FLS2-dependent immunity, flagellin in the cytosol does not elicit immune-associated cell death. We postulate that a death response to translocated MAMPs would produce vulnerability to the many necrotrophic pathogens of plants, such as P. carotovorum, which differ from P. syringae and other (hemi)biotrophic pathogens in benefitting from death-associated immune responses.

CsrA-FliW interaction governs flagellin homeostasis and a checkpoint on flagellar morphogenesis in Bacillus subtilis

CsrA is a widely distributed RNA binding protein that regulates translation initiation and/or mRNA stability of target transcripts. CsrA activity is antagonized by sRNA(s) containing multiple CsrA binding sites in several Gram-negative bacterial species. Here we discover FliW, the first protein antagonist of CsrA activity that constitutes a partner switching mechanism to control flagellin synthesis in the Gram-positive organism Bacillus subtilis. Following the flagellar assembly checkpoint of hook completion, secretion of flagellin (Hag) releases FliW protein from a FliW-Hag complex. FliW then binds to CsrA and relieves CsrA-mediated translational repression of hag for flagellin synthesis concurrent with filament assembly. Thus, flagellin homeostatically restricts its own translation. Homeostatic autoregulation may be a general mechanism to precisely control structural subunits required at specific times and in finite amounts such as those involved in the assembly of flagella, type III secretion machines and pili. Finally, phylogenetic analysis suggests that CsrA, a highly pleiotropic virulence regulator in many bacterial pathogens, had an ancestral role in flagellar assembly and evolved to co-regulate various cellular processes with motility.

The physiologic and phenotypic alterations due to macrolide exposure in Campylobacter jejuni

Physiologic and phenotypic alterations in the context of antibiotic resistance have been extensively studied in some bacteria. However there are not enough data addressing these alterations due to macrolide resistance in Campylobacter jejuni. The present study examined the fitness cost imposed by different macrolide resistance mutations and the phenotypic alterations due to exposure to macrolides in C. jejuni. C. jejuni was induced with different macrolide agents to obtain different macrolide resistance mutations. The results revealed that the mutations significantly imposed defect variations on the doubling time and the relative fitness in the resistant strains when competed against the susceptible strain. Furthermore macrolides through induction or exposure to sub-MIC concentrations impaired the motility of C. jejuni in 0.4% MH agar plates. Electron microscope analysis revealed the absence of flagellar filaments from strains exposed to macrolides. SDS-PAGE demonstrated that macrolides have no effect on protein synthesis and immunoblotting analysis further confirmed that flagellin was fully synthesized within C. jejuni strains exposed to macrolides. Nevertheless C. jejuni strains exposed to macrolides demonstrated defect in their excreted flagellin into the supernatant compared to strains not exposed to macrolides. Accordingly we speculated that macrolides inhibited flagellar filament formation in the strains exposed to macrolides via affecting the secretion of flagellin without affecting the amount synthesized within the bacteria. Taken together these findings demonstrated that different macrolide resistance mutations imposed different fitness costs and exposure to macrolides resulted in phenotypic alterations such as inhibition of flagellar filament formation and loss of motility in C. jejuni.

FlhA provides the adaptor for coordinated delivery of late flagella building blocks to the type III secretion system

Flagella are the bacterial organelles of motility and can play important roles in pathogenesis. Flagella biosynthesis requires the coordinated export of huge protein amounts from the cytosol to the nascent flagellar structure at the cell surface and employs a type III secretion system (T3SS). Here we show that the integral membrane protein FlhA from the gram-positive bacterium Bacillus subtilis acts as an adaptor for late export substrates at the T3SS. The major filament protein (flagellin) and the filament-cap protein (FliD) bind to the FlhA cytoplasmic domain (FlhA-C) only in complex with their cognate chaperones (FliS and FliT). To understand the molecular details of these interactions we determined the FlhA-C crystal structure at 2.3 A resolution. FlhA-C consists of an N-terminal linker region, three subdomains with a novel fold, and a disordered region essential for the adaptor function. We show that the export protein FliJ associates with the linker region and modulates the binding properties of FlhA-C. While the interaction of FliD/FliT is enhanced, flagellin/FliS is not affected. FliJ also keeps FliT associated with FlhA-C and excess of FliT inhibits binding of FliD/FliT, suggesting that empty FliT chaperones stay associated with FliJ after export of FliD. Taken together, these results allow to propose a model that explains how the T3SS may switch from the stoichiometric export of FliD to the high-throughput secretion of flagellin.

Secretion of flagellin by the LEE-encoded type III secretion system of enteropathogenic Escherichia coli

BackgroundEnteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) pathogen that possesses a type III secretion system (T3SS) encoded within the locus of enterocyte effacement (LEE). The LEE is essential for A/E lesion formation and directs the secretion and translocation of multiple LEE-encoded and non-LEE encoded effector proteins into the cytosol of infected cells. In this study we used proteomics to compare proteins exported to the culture supernatant by wild type EPEC E2348/69, a ΔespADB mutant and a ΔescF T3SS mutant.ResultsWe observed that flagellin was consistently and strongly present in the secretome of wild type EPEC and the ΔespADB mutant but present only weakly in the secretome of the ΔescF mutant. Given the ancestral relationship between the flagella export apparatus and virulence associated T3SSs, we investigated whether FliC could utilise the LEE-encoded T3SS for export. In the absence of a functional flagella export apparatus, we showed that FliC could be secreted by the LEE-encoded T3SS and stimulate (Toll-like receptor 5) TLR5 signalling but could not confer motility.ConclusionSince the secretion of FliC during A/E lesion formation would presumably be disadvantageous for the pathogen, we propose that virulence associated T3SSs and flagella T3SSs have evolved through a system of chaperones and complex regulatory pathways to be functional at different times to ensure that FliC secretion does not occur during T3SS effector translocation.

The Salmonella enterica serotype Typhi regulator TviA reduces interleukin-8 production in intestinal epithelial cells by repressing flagellin secretion

Unlike non-typhoidal Salmonella serotypes, S. enterica serotype Typhi does not elicit neutrophilic infiltrates in the human intestinal mucosa. The Vi capsule-encoding tviABCDEvexABCDE operon (viaB locus) is a S. Typhi-specific DNA region preventing production of interleukin (IL)-8 during infection of intestinal epithelial cells. We elucidated the mechanism by which the viaB locus reduces IL-8 production in human colonic epithelial (T84) cells. A S. Typhi tviABCDEvexABCDE deletion mutant, but not a tviBCDEvexABCDE deletion mutant, elicited increased IL-8 production, which could be reduced to wild-type levels by introducing the cloned tviA regulatory gene. Thus, IL-8 expression in T84 cells was modulated by the TviA regulatory protein, but not by the Vi capsular antigen. Consistent with previous reports, IL-8 secretion by T84 cells was dependent on the presence of the flagellin protein FliC. TviA reduced expression of flhDC::lacZ and fliC::lacZ transcriptional fusions and secretion of FliC in S. Typhi. Introduction of tviA into S. enterica serotype Typhimurium reduced flagellin secretion and IL-8 expression. In conclusion, the viaB locus reduces IL-8 production in T84 cells by a TviA-mediated repression of flagellin secretion. Our data suggest that changes in flagella gene regulation played an important role during evolution of the human-adapted S. Typhi.

Bacteria Respond to Host Cell Lysophospholipids

Evaluation of: Subramanian N, Qadri A.: Lysophospholipid sensing triggers secretion of flagellin from pathogenic Salmonella. Nat. Immunol. 7(6), 583–589 (2006). The ability of host cells to detect pathogen-specific molecules has now become readily appreciated. Toll-like receptors can detect conserved structures on pathogens and once recognized can trigger an innate immune response. In this report, Subramanian and Qadri present evidence that the host does not simply respond to the presence of bacteria or bacterial factors, but can also trick pathogens into revealing themselves by inducing the secretion of one of these pathogen-associated molecules.

Flagellin, a Key Immune Trigger

The intracellular pattern recognition receptor Ipaf, which is a member of the NLR (for nucleotide-binding oligomerization domain-leucine-rich repeat or NACHT-leucine-rich repeat receptor) family, is involved in caspase-1 activation, which can stimulate interleukin-1β (IL-1β) secretion or lead to apoptosis, in response to bacterial infection of macrophages. Miao et al . and Franchi et al . both show that caspase-1 activation and IL-1β secretion by macrophages infected with Salmonella typhimurium required the bacterial type III secretion system (TTSS) and the flagellar protein flagellin, of which there are two variants, FliC and FliB. Bacteria lacking FliC and FliB or a functional TTSS did not stimulate caspase-1 activation or IL-1β secretion. Application of flagellin extracellularly did not trigger a macrophage response (because mouse macrophages lack the Toll-like receptor TLR5 that allows recognition of extracellular flagellin). But introduction of flagellin into the cytoplasm of macrophages using agents that mildly disrupt the membrane triggered IL-1β secretion; however, this was not observed in macrophages lacking Ipaf. Thus, the bacteria appear to release flagellin through the TTSS into the host cytoplasm, where it is recognized by Ipaf, which stimulates the inflammatory response. Subramanian and Qadri showed that exposure of Salmonella to intestinal epithelial cells (IECs) stimulated the secretion of monomeric flagellin into the culture medium, which was then able to stimulate IL-8 production in uninfected IECs when the flagellin-containing culture medium was applied to IECs (which have TLR5 and can respond to extracellular flagellin). CaCo-2 cells, an IEC cell line, exposed to various treatments to denature or inactivate proteins, were still capable of stimulating flagellin secretion by Salmonella ; thus, lipid mediators were tested. Exposure of Salmonella to lysophosphatidic acid (LPA) or lysophosphatidylcholine (LPC) stimulated flagellin secretion. Lysophospholipids are released by cells through the activity of phospholipase A 2 (PLA 2 ), and inhibition of two types of PLA 2 suppressed the stimulation of flagellin secretion from the bacteria in response to exposure to IECs or culture medium from IECs. In vivo, inhibition of calcium-independent cell-associated PLA 2 inhibited flagellin release by Salmonella in infected mice. Secretion of flagellin in response to lysophospholipids involved de novo protein synthesis, and LPC and LPA both stimulated a reporter gene driven by the fliC promoter, suggesting that lysophospholipids may trigger the adenosine 3′,5′-monophosphate (cAMP) signaling pathway that activates flagellar biosynthesis. Exposure of Salmonella to glucose, which decreases cAMP concentrations, reduced flagellin secretion in response to LPC. Roy and Zamboni discuss the implications of these three articles and propose that host cells trigger flagellin production and secretion by the bacteria, effectively "'tricking' the bacteria into revealing themselves to host flagellin sensors." E. A. Miao, C. M. Alpuche-Aranda, M. Dors, A. E. Clark, M. W. Bader, S. I. Miller, A. Aderem, Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1β via Ipaf. Nat. Immunol. 7 , 569-575 (2006). [PubMed] L. Franchi, A. Amer, M. Body-Malapel, T.-D. Kanneganti, N. Özören, R. Jagirdar, N. Inohara, P. Vandenabeele, J. Bertin, A. Coyle, E. P. Grant, G. Núñez, Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1β in salmonella-infected macrophages. Nat. Immunol. 7 , 576-582 (2006). [PubMed] N. Subramanian, A. Qadri, Lysophospholipid sensing triggers secretion of flagellin from pathogenic salmonella. Nat. Immunol. 7 , 583-589 (2006). [PubMed] C. R. Roy, D. S. Zamboni, Cytosolic detection of flagellin: A deadly twist. Nat. Immunol. 7 , 549-551 (2006). [PubMed]

Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1β via Ipaf

Macrophages respond to Salmonella typhimurium infection via Ipaf, a NACHT-leucine-rich repeat family member that activates caspase-1 and secretion of interleukin 1beta. However, the specific microbial salmonella-derived agonist responsible for activating Ipaf is unknown. We show here that cytosolic bacterial flagellin activated caspase-1 through Ipaf but was independent of Toll-like receptor 5, a known flagellin sensor. Stimulation of the Ipaf pathway in macrophages after infection required a functional salmonella pathogenicity island 1 type III secretion system but not the flagellar type III secretion system; furthermore, Ipaf activation could be recapitulated by the introduction of purified flagellin directly into the cytoplasm. These observations raise the possibility that the salmonella pathogenicity island 1 type III secretion system cannot completely exclude 'promiscuous' secretion of flagellin and that the host capitalizes on this 'error' by activating a potent host-defense pathway.

Lysophospholipid sensing triggers secretion of flagellin from pathogenic salmonella

Flagellin induces inflammatory and innate immune responses through activation of Toll-like receptor 5. Here we show that proinflammatory monomeric flagellin produced by salmonella during infection of intestinal epithelial cells was not derived from polymeric bacterial cell wall-associated flagellum but instead was synthesized and secreted de novo by the bacterium after direct sensing of host-produced lysophospholipids. Inhibition of lysophospholipid biosynthesis in intestinal epithelial cells reduced flagellin production and release from salmonella. Lysophospholipids induced a cAMP-dependent signaling pathway in salmonella that resulted in production and secretion of active flagellin. The induction of Toll-like receptor ligand synthesis and secretion by a host signal represents a previously unknown regulatory mechanism for inflammation and innate immunity during infection with a bacterial pathogen.

Flagellin from Pseudomonas syringae pv. tabaci induced hrp-independent HR in tomato

We have previously shown that flagellin of Pseudomonas syringae pv. tabaci is an elicitor that induces a hypersensitive reaction (HR) in nonhost tomato cells. Flagellin is the major HR elicitor produced by this pathogen, as shown by the inability of a flagellin-defective mutant, ΔfliC, to induce HR. Also, a ΔfliD mutant that secretes large amounts of monomer flagellins induces a strong HR in tomato. In this study, the possible involvement of an Hrp type III secretion system (TTSS) in flagellin-induced HR was investigated using flagella-defective mutants or Hrp TTSS-defective mutants. The hrcC gene encodes HrcC protein, which is required for Hrp pilus formation in the outer membrane. An hrcC mutation, introduced into the wild-type, ΔfliC, and ΔfliD mutants of P. syringae pv. tabaci did not affect swimming motility or flagellin secretion, whereas all ΔhrcC, ΔfliC, and ΔfliD mutants lost the ability to cause disease on host tobacco leaves. However, the ΔhrcC mutant and the ΔfliD/ΔhrcC double mutant were still able to induce HR cell death, expression of one of the defense-related genes hsr203J, and the generation of hydrogen peroxide in nonhost tomato cells. Thus, flagellin is required for both pathogenicity in host tobacco and HR in nonhost tomato. On the other hand, hrp TTSS is necessary for pathogenicity on host tobacco but is not indispensable to induce HR in nonhost tomato. These results clearly show that flagellin-induced HR is hrp-independent in tomato.

Mutants in flaI and flaJ of the archaeon Methanococcus voltae are deficient in flagellum assembly.

The fla gene locus of Methanococcus voltae encodes the major structural components of the flagellum as well as other flagellar-related proteins. The flaHIJ genes have been found in all flagellated archaea, suggesting a central role in flagella biogenesis. FlaI shares similarity with the type II and type IV secretion NTPases (such as PilB, VirB11 and TadA), and FlaJ exhibits similarity to putative bacterial integral membrane proteins involved in type IV pilus biogenesis such as TadB. In this study, reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting data revealed that flaHIJ are co-transcribed with the upstream structural flagellin genes, thus demonstrating the expression of the entire fla gene cluster in vivo. Non-polar mutants in flaI and flaJ of M. voltae were isolated using insertional inactivation via a novel mutagenic vector. These mutants were non-motile and non-flagellated by microscopy, demonstrating the involvement of FlaI and FlaJ in flagella biogenesis. Interestingly, all the mutants maintained the ability to produce and localize flagellins to the cytoplasmic membrane. Amino-terminal sequencing of flagellins produced by the flaJ mutant strain revealed that the flagellins did not have their cognate leader peptides, thus indicating that preflagellin processing had occurred in vivo. This result was confirmed using an in vitro processing assay. The fla- phenotype and protein secretion characteristics of the flaI and flaJ mutants therefore implicate these respective genes in archaeal flagellin secretion and assembly. These findings further support a model describing the archaeal flagellum as a novel prokaryotic motility structure.

The Salmonella typhimurium Flagellar Basal Body Protein FliE Is Required for Flagellin Production and to Induce a Proinflammatory Response in Epithelial Cells

During apical colonization by Salmonella typhimurium, intestinal epithelial cells orchestrate a proinflammatory response that involves secretion of chemoattractants, predominantly interleukin-8, which coordinate neutrophil trans-epithelial migration at the site of infection. This host-pathogen interaction requires several S. typhimurium genes. To identify novel genes that participate in this pathogen-induced proinflammatory response, we created S. typhimurium Tn-10 transposon mutants and identified a single mutant with Tn-10 insertional inactivation within the fliE flagellar locus that was able to adhere to and invade intestinal epithelial cells normally but was unable to induce interleukin-8 secretion in host cells. The fliE-deficient mutant failed to secrete flagellin and lacked any surface assembly of flagellae. Unlike wild-type S. typhimurium, the fliE-deficient mutant did not activate the IkappaBalpha/NF-kappaB signaling pathway or induce the coordinated trans-epithelial migration of isolated human neutrophils. Transcomplementation of the fliE-deficient mutant with a wild-type fliE-harboring plasmid restored all defects and produced a wild-type S. typhimurium phenotype. Furthermore, functional down-regulation of basolateral TLR5 completely inhibited the monolayers' ability to respond to both wild-type S. typhimurium and purified flagellin but had no affect on tumor necrosis factor alpha-induced responses. We therefore conclude that S. typhimurium fliE is essential for flagellin secretion, flagellar assembly, and S. typhimurium-induced proinflammatory responses through basolateral TLR5 and is consistent with the emerging model of S. typhimurium flagellin-induced inflammation.

Characterization of Flagellum Gene Families of Methanogenic Archaea and Localization of Novel Flagellum Accessory Proteins

Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii has been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes, flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes of M. voltae were successfully overexpressed as His-tagged proteins in Escherichia coli. These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionated M. voltae cells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly, flaD was found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes of M. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.

Insertional inactivation of the flaH gene in the archaeon Methanococcus voltae results in non-flagellated cells

The marine methanogen Methanococcus voltae possesses two transcriptional units that encode a total of four flagellins. Immediately downstream of the flagellin genes are a number of ORFs, some of which are cotranscribed with the flagellin genes. These putative genes have been named flaCDEFGHIJ, although no biochemical data has implicated them in flagellar morphogenesis. None of the flaC-J genes has homology to any bacterial gene, with the exception of flaI, which shows homology to pilT, a gene that encodes a nucleotide binding protein of the type IV pilus family. In this study, insertional mutations in flaH of M. voltae were identified. The mutants were non-motile and non-flagellated as determined by electron microscopy. Southern hybridization experiments confirmed the insertion of a mutagenic vector into flaH and indicated that two, tandem, copies of the vector were present. It is believed that insertion of the vector into flaH should disrupt the transcription of flaIJ due to polar effects. The flaH mutant displayed the same pattern of multiple mRNA transcripts, all originating upstream of flaB1, as the wild-type cells. Northern hybridization experiments failed to detect a flaHIJ transcript in either wild-type or mutant cells. Immunoblotting experiments indicated, however, that the mutants produced similar amounts of flagellin, FlaD and FlaE to wild-type cells. Flagellin localization experiments suggest that the flaH mutant is deficient in flagellin secretion and/or assembly. The mutant also displayed similar preflagellin peptidase activity to the wild-type cells, indicating that none of the genes flaHIJ is likely to be the gene that encodes this enzyme, which is required for cleaving the leader peptide from the preflagellins prior to their incorporation into the flagellar filament. This is the first data indicating that the flaHIJ gene cluster is essential for flagellation in methanogens.

FliU and fliV: two flagellar genes essential for biosynthesis of Salmonella and Escherichia coli flagella

The possible functions of two recently described flagellar genes, fliU and fliV, have been examined. Introduction of gene fliC, encoding the bacterial flagellin protein, into a number of flagellin-deficient Salmonella and Escherichia coli strains failed to complement the mutations in these strains, and the FliC flagellin was accumulated in the bacterial cytoplasm. Complementation with fliU and fliV, which map downstream of fliC, restored motility to some of the mutants which became flagellated. After inactivation of either fliU or fliV, such complementation no longer occurred and the flagellin protein accumulated in the cytoplasm, which suggested that both genes are required for the secretion of flagellin and expression of motility. Expression of these genes from high copy number plasmids resulted in the synthesis of exceptionally long flagella and in detection of the FliV protein on polyacrylamide gels.