• Molecules and Cells
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• v.43 no.3
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• pp.251-263
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• 2020

Flagellin, a major structural protein of the flagellum found in all motile bacteria, activates the TLR5- or NLRC4 inflammasome-dependent signaling pathway to induce innate immune responses. Flagellin can also serve as a specific antigen for the adaptive immune system and stimulate anti-flagellin antibody responses. Failure to recognize commensal-derived flagellin in TLR5-deficient mice leads to the reduction in anti-flagellin IgA antibodies at steady state and causes microbial dysbiosis and mucosal barrier breach by flagellated bacteria to promote chronic intestinal inflammation. Despite the important role of anti-flagellin antibodies in maintaining the intestinal homeostasis, regulatory mechanisms underlying the flagellin-specific antibody responses are not well understood. In this study, we show that flagellin induces interferon-β (IFN-β) production and subsequently activates type I IFN receptor signaling in a TLR5- and MyD88-dependent manner in vitro and in vivo. Internalization of TLR5 from the plasma membrane to the acidic environment of endolysosomes was required for the production of IFN-β, but not for other pro-inflammatory cytokines. In addition, we found that anti-flagellin IgG2c and IgA responses were severely impaired in interferon-alpha receptor 1 (IFNAR1)-deficient mice, suggesting that IFN-β produced by the flagellin stimulation regulates anti-flagellin antibody class switching. Our findings shed a new light on the regulation of flagellin-mediated immune activation and may help find new strategies to promote the intestinal health and develop mucosal vaccines.

• Journal of Microbiology and Biotechnology
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• v.22 no.7
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• pp.907-916
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• 2012

Burkholderia cepacia is an important pathogen that often causes pneumonia in immunocompromised individuals. Here, it was demonstrated that the TLR5 agonist flagellin could locally activate innate immunity. This was characterized by rapid expressions of IL-$1{\beta}$, TNF-${\alpha}$, and iNOS mRNA and a delay in the expression of IL-10 mRNA. A significant elevation in the IL-$1{\beta}$, TNF-${\alpha}$, and nitric oxide levels was also noted. In the respiratory tract, flagellin induced neutrophil infiltration into the airways, which was observed by histopathological examination and confirmed by the neutrophil count and level of myeloperoxidase activity. This was concomitant with a high activity of alveolar macrophages that engulfed and killed B. cepacia in vitro. The flagellin mucosal treatment improved the B. cepacia clearance in the mouse lung. Thus, the present findings illustrate the profound stimulatory effect of flagellin on the lung mucosal innate immunity, a response that needs to be exploited therapeutically to prevent the development of respiratory tract infection by B. cepacia.

• Journal of Life Science
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• v.23 no.2
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• pp.299-305
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• 2013

Paenibacillus is a gram-positive, spore-forming aerobes that was previously classified as a Bacillus species. Paenibacillus sp. CK214 was highly motile on LB agar plates and showed typical colonial morphology of Paenibacillus. However, its motility was defective in the absence of glucose. Electron microscopic observation revealed that the cells of CK214 cultured on LB agar plates were peritrichously flagellated but not flagellated in the presence of glucose. Flagellar filaments were purified by centrifugation after shearing off from the CK214 cells with vigorous pipetting. The purified protein was composed of a single flagellin with an apparent molecular size of 29 kDa. Recognition of the protein by anti-Edwardsiella tarda flagellin protein antibody demonstrates that the protein is a flagellin protein. A decreased level of flagellin protein was detected in CK214 cells grown under glucose-supplemented media.

• Korean Journal of Veterinary Service
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• v.37 no.3
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• pp.157-164
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• 2014

Clostridium chauvoei is the etiologic agent of blackleg, a high mortality rated disease infection mainly cattle. In the present study, the partial sequences of 16S rRNA and flagellin gene of C. chauvoei isolated in Jeonbuk, Korea were determined and compared with those of reference strain. Oligonucleotide primers were designed to amplify a 811 bp fragment of 16S rRNA gene and 1229 bp fragment of flagellin gene. Sequencing analysis of 16S rRNA gene showed high homology to the reference strains ranging 82.3% to 100%, while flagellin gene were different from published foreign clostridia, showing 98.7% to 72.0% nucleotide sequence homology. Phylogenetic analysis based on 16S rRNA gene revealed the close phylogenetic relationship of C. chauvoei and C. septicum in cluster I, which includes C. carnis, C. tertium, C. quinii, C. celatum, C. perfringens, C. absonum, C. botulinum B. Phylogentic analysis also revealed that flagellin gene formed a single cluster with C. chauvoei, C. septicum, C. novyi A, C. novyi B, C. tyrobutylicum, C. acetobutylicum. The genetic informations obtained from this study could be useful for the molecular study of C. chauvoei.

• Journal of Life Science
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• v.21 no.10
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• pp.1487-1493
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• 2011

E. tarda, a fish pathogen, can survive in seawater under relatively high salt conditions as well as in fish under physiological salt conditions. Bacterial growth under different salt concentrations may influence the expression of genes involved in bacterial structure and physiology. The growth rate of E. tarda culture in high salt (3.5% NaCl) was similar to that in low salt (1.0% NaCl, physiological salt concentration). Interestingly, the strain moved much faster in low salt conditions than in high salt conditions. Electron microscopic observation demonstrated that the bacterial cells grown in high salt had less or no flagellation. Obvious flagellation was observed in the parental strain E. tarda CK41 grown in low-salt condition. Two putative genes coding flagellin were identified in the E. tarda genome sequences. The amino acid sequence comparison of each gene revealed 93% identities. A flagellin gene was PCR amplified and cloned into a cloning vector. Using an E. coli protein expression system, a part of flagellin protein was overexpressed. Using the purified protein, an anti-flagellin antibody was raised in the rabbit. Immunoblot analyses with flagellin specific antibody demonstrated that E. tarda CK41 expressed falgellin in low salt conditions, which is consistent with the results seen in motility assay and microscopic observation. This is the first report of salt regulated flagella expression in E. tarda.

• Korean Journal of Microbiology
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• v.50 no.4
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• pp.281-284
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• 2014

Immune defense responses against Pseudomonas aeruginosa infection play an important role in maintaining homeostasis in the human body. Previously, we reported that expression of the bradykinin receptor (BR) is induced in response to P. aeruginosa infection. However, the factors responsible for the induction was uncertain. Here, we found that the type III secretion system (T3SS) is responsible for the induction of BR expression, and nucleoside diphosphate kinase (Ndk), as a novel T3SS effector, mediates the upregulation. The Ndk-mediated expression of BR was not induced by fliC mutant treatment, indicating the involvement of flagellin, one of the well-known pathogen-associated molecular patterns (PAMPs). Taken together, this study demonstrated that Ndk cooperates with flagella in the development of defense responses against P. aeruginosa infection.

• Animal Bioscience
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• v.36 no.2
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• pp.315-321
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• 2023

Objective: The study was conducted to investigate the dephosphorylation of Pseudomonas aeruginosa flagellin (PA FLA) by sweet potato purple acid phosphatase (PAP) and the effect of the enzyme on the flagellin-mediated inflammatory response in the A549 lung epithelial cell line. Methods: The activity of sweet potato PAP on PA FLA was assayed at different pH (4, 5.5, 7, and 7.5) and temperature (25℃, 37℃, and 55℃) conditions. The release of interleukin-8 (IL-8) and the activation of nuclear factor kappa- light-chain-enhancer of activated B cells (NF-κB) in A549 cells exposed to PA FLA treated with or without sweet potato PAP was measured using IL-8 and NF-κB ELISA kits, respectively. The activation of toll-like receptor 5 (TLR5) in TLR5-overexpressing HEK-293 cells exposed to PA FLA treated with or without sweet potato PAP was determined by the secreted alkaline phosphatase-based assay. Results: The dephosphorylation of PA FLA by sweet potato PAP was favorable at pH 4 and 5.5 and highest at 55℃. PA-FLA treated with the enzyme decreased IL-8 release from A549 cells to about 3.5-fold compared to intact PA FLA at 1,000 ng/mL of substrate. Moreover, PA-FLA dephosphorylated by the enzyme repressed the activation of NF-κB in the cells compared to intact PA FLA. The activation of TLR5 by PA-FLA was highest in TLR-overexpressing HEK293 cells at a substrate concentration of 5,000 ng/mL, whereas PA FLA treated with the enzyme strongly repressed the activation of TLR5. Conclusion: Sweet potato PAP has the potential to be a new alternative agent against the increased antibiotic resistance of P. aeruginosa and may be a new conceptual feed additive to control unwanted inflammatory responses caused by bacterial infections in animal husbandry.

• The Journal of the Korean Society for Microbiology
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• v.35 no.5_6
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• pp.365-365
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• 2000

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1227-1231
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• 2004

Campylobacter, one of the emerging foodborne pathogens, is highly adaptable to the external environments by changing its morphology. In the present study, a question of whether the whole-cell antibody would still be effective for its detection even though the morphology of C. jejuni was changed was examined. When microaerophilic C. jejuni was exposed to aerobic conditions for 48 h, its morphological change was detected by confocal laser scanning microscope: Its morphology was confirmed as a spiral-bacilli form in microaerobic condition, however, as a coccoid form with a little spiral-bacilli form, when exposed to aerobic conditions. Also, the expressions of the whole-cell proteins of C. jejuni, and the suppression or induction of newly synthesized proteins in both aerobic and microaerobic conditions were analyzed by two dimensional gel electrophoresis. Additionally, immunoblotting assay with the whole cell antibody for the proteins expressed under the two conditions was performed. It was confirmed that the commercial whole-cell antibody of C. jejuni raised in rabbit was reactive. When analyzed with MALDI- TOF MS, the expressed proteins were confirmed as flagellins. Therefore, even though the morphology changed in aerobic condition, these flagellins were expressed and worked as the eitope proteins, thus making it possible to utilize for the development of an immunosensor for real-time detection of any kind of C. jejuni cell.

• Journal of Veterinary Science
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• v.25 no.1
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• pp.4.1-4.14
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• 2024

Background: Lawsonia intracellularis is the causative agent of proliferative enteropathy and is associated with several outbreaks, causing substantial economic loss to the porcine industry. Objectives: In this study, we focused on demonstrating the protective effect in the mouse model through the immunological bases of two vaccine strains against porcine proliferative enteritis. Methods: We used live-attenuated Salmonella Typhimurium (ST) secreting two selected immunogenic LI antigens (Lawsonia autotransporter A epitopes and flagellin [FliC]-peptidoglycan-associated lipoprotein-FliC) as the vaccine carrier. The constructs were cloned into a Salmonella expression vector (pJHL65) and transformed into the ST strain (JOL912). The expression of immunogenic proteins within Salmonella was evaluated via immunoblotting. Results: Immunizing BALB/c mice orally and subcutaneously induced high levels of LI-specific systemic immunoglobulin G and mucosal secretory immunoglobulin A. In immunized mice, there was significant upregulation of interferon-γ and interleukin-4 cytokine mRNA and an increase in the subpopulations of cluster of differentiation (CD) 4⁺ and CD 8⁺ T lymphocytes upon splenocytes re-stimulation with LI antigens. We observed significant protection in C57BL/6 mice against challenge with 10^6.9 times the median tissue culture infectious dose of LI or 2 × 10⁹ colony-forming units of the virulent ST strain. Immunizing mice with either individual vaccine strains or co-mixture inhibited bacterial proliferation, with a marked reduction in the percentage of mice shedding Lawsonia in their feces. Conclusions: Salmonella-mediated LI gene delivery induces robust humoral and cellular immune reactions, leading to significant protection against LI and salmonellosis.