<i>Salmonella</i> Flagellin-Dependent Proinflammatory Responses Are Localized to the Conserved Amino and Carboxyl Regions of the Protein

Eaves-Pyles, Tonyia; Wong, Hector R.; Odoms, Kelli; Pyles, Richard B.

doi:10.4049/jimmunol.167.12.7009

Cited by 125 publications

(111 citation statements)

References 27 publications

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“…Although the TLR5 dependence of Gram-negative flagellin signaling has been well documented, the structural requirements for the interaction between flagellin and TLR5 are only partially understood. Structure/function studies with soluble flagellin indicate that the amino and carboxyl constant regions are most critical for TLR5 signaling (22,23). We have shown that flagellin is active at concentrations in the picomolar to nanomolar range (24), a finding that is consistent with a very high affinity interaction.…”

supporting

confidence: 68%

“…The results of two recent studies (22,23) indicate that the conserved amino and carboxyl regions of flagellin are required for biologic activity. This appears to be in conflict with our observation (24) that a peptide containing only these two regions was inactive.…”

Section: Discussionmentioning

confidence: 99%

“…This appears to be in conflict with our observation (24) that a peptide containing only these two regions was inactive. However, as noted by Eaves-Pyles et al (23), such a peptide may not function because it lacks a bridge that facilitates the necessary interaction between the two ends of the flagellin protein. The involvement of the highly conserved termini of flagellin in TLR5 binding is consistent with the notion that a key feature of pathogen-associated molecular patterns is their invariance between microorganisms of a given class (32).…”

Section: Discussionmentioning

confidence: 99%

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Identification of a Sequence in Human Toll-like Receptor 5 Required for the Binding of Gram-negative Flagellin

Mizel¹,

West²,

Hantgan

2003

Journal of Biological Chemistry

136

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Activation of the innate immune response by bacteria is mediated by a group of molecules that have collectively been termed modulins (1). This family of molecules includes a wide variety of substances including lipopolysaccharide (LPS) 1 from Gram-negative bacteria, lipoteichoic acid from Gram-positive bacteria, several different viral envelope glycoproteins, and flagellins from Gram-negative bacteria. Monocytes, macrophages, neutrophils, and epithelial cells respond to modulins by producing proinflammatory mediators such as tumor necrosis factor ␣, interleukin-1␤, and nitric oxide. Many bacterial modulins signal via toll-like receptors (TLR), a family of cell surface molecules that possess a leucine-rich extracellular domain and a cytoplasmic toll/interleukin-1 receptor homology domain (2).Although substantial progress has been made in our understanding of the intracellular pathways involved in TLR signaling, we have very little knowledge of the factors that determine the specificity of individual TLRs. For example, the common structural features that allow a diverse group of agonists such as LPS (3-5), heat shock protein 60 (6), respiratory syncytial virus F protein (7), and Escherichia coli P fimbriae (8) to signal via TLR4 are not readily apparent. Although investigators have accepted the notion that TLRs recognize pathogen-associated molecular patterns (9), we have a limited understanding of the structural elements within TLRs that determine their selectivity for specific agonists. The interaction of LPS with CD14 and TLR4 has been the most extensively studied system. LPS binding to CD14 appears to be required for the subsequent interaction of LPS with TLR4 (10) and MD-2, a TLR4-associated protein that may facilitate or enhance the cell surface expression of TLR4 (11). Difference in LPS structure may influence the quality of these interactions (12). Although the region of TLR4 that is involved in the recognition of LPS has not been defined, the structural features of CD14 that are involved in the recognition of LPS have been well characterized (13-16). In a recent study, it was suggested that peptidoglycan from Staphylococcus aureus can bind directly to the extracellular domain of TLR2 (17). However, soluble CD14 was found to enhance this interaction, leaving open the possibility that CD14 or similar proteins might be mediators of the interaction between peptidoglycan and TLR2.Recent reports from our laboratory and others have demonstrated that flagellins from Gram-negative bacteria activate a range of inflammatory cells via a TLR5-dependent signaling pathway (18 -20). As with other TLRs, flagellin signaling via TLR5 involves the activation of the interleukin-1 receptorassociated kinase (20,21). Although the TLR5 dependence of Gram-negative flagellin signaling has been well documented, the structural requirements for the interaction between flagellin and TLR5 are only partially understood. Structure/function studies with soluble flagellin indicate that the amino and carboxyl constant regions are most critic...

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supporting

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Identification of a Sequence in Human Toll-like Receptor 5 Required for the Binding of Gram-negative Flagellin

Mizel¹,

West²,

Hantgan

2003

Journal of Biological Chemistry

136

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“…Furthermore, the motif 89 -96 is absolutely required (8 -10). The middle (outer) domain of flagellin FliC comprises amino acids from positions 170 -400 and is not mandatory for TLR5 signaling (9,10). It is designated as a hypervariable region, since the primary sequences greatly vary in composition and size from one bacterial species to another.…”

Section: Deletion Of Flagellin's Hypervariable Region Abrogates Antibmentioning

confidence: 99%

Deletion of Flagellin’s Hypervariable Region Abrogates Antibody-Mediated Neutralization and Systemic Activation of TLR5-Dependent Immunity

Nempont

Cayet

Rumbo

et al. 2008

The Journal of Immunology

115

134

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TLRs trigger immunity by detecting microbe-associated molecular patterns (MAMPs). Flagellin is a unique MAMP because it harbors 1) an antigenic hypervariable region and 2) a conserved domain involved in TLR5-dependent systemic and mucosal proinflammatory and adjuvant activities. In this study, the contribution of the flagellin domains in TLR5 activation was investigated. We showed that TLR5 signaling can be neutralized in vivo by flagellin-specific Abs, which target the conserved domain. However, deletions of flagellin’s hypervariable region abrogated the protein’s intrinsic ability to trigger the production of neutralizing Abs. The fact that MAMP-specific Abs block TLR-mediated responses shows that this type of neutralization is a novel mechanism for down-regulating innate immunity. The stimulation of mucosal innate immunity and adjuvancy to foreign Ag was not altered by the hypervariable domain deletions. In contrast, this domain is essential to trigger systemic innate immunity, suggesting that there are distinct mechanisms for TLR5 activation in systemic and mucosal compartments. In summary, specific MAMP determinants control the production of neutralizing Abs and the compartmentalization of innate responses.

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“…Recombinant flagellin of B. pseudomallei was obtained from E. coli BL21 (pGex4T-2/fliC) and the flagellin preparation was then digested with thrombin as described elsewhere (4). Any contaminating lipopolysaccharide (LPS) present in the flagellin was removed using a polymyxin B column (8) in accordance with the manufacturer's instructions (Detoxi-gel; Rockford, Ill., U.S.A.). This was repeated until the concentration of LPS was less than 1 pg/ml as determined by the Limulus Amebocyte Lysate assay (BioWhittaker, Inc., Walkersville, Md., U.S.A.).…”

Section: Methodsmentioning

confidence: 99%

Immunostimulatory Flagellin from Burkholderia pseudomallei Effects on an Increase in the Intracellular Calcium Concentration and Up‐Regulation of TNF‐α by Mononuclear Cells

Chen

Lin

et al. 2007

Microbiology and Immunology

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Using flow cytometry analysis, the flagellin of Burkholderia pseudomallei acts as a signalling inducer, and evokes an increase in the intracellular calcium ion concentration ([Ca2+]i) in human peripheral blood mononuclear cells (PBMC). The cells with increased [Ca2+]i segregate into the live monocyte gate and not into the live lymphocyte gates. The stimulated [Ca2+]i increase can be neutralized with anti‐flagellin antibodies. In the absence of [Ca2+], [Ca2+]i was increased rapidly in flagellin‐treated cells compared to non‐flagellin‐treated cells only after the addition of 1 mM CaCl2. Selective calcium antagonists were used to effectively block the [Ca2+]i signal, revealing that this signal was decreased by the addition of L‐type calcium channel blockers (diltiazem, nifedipine and verapamil) and La2+ but was not changed by the addition of a T‐type calcium channel blocker (flunarizine). It seemed that flagellin facilitates [Ca2+]i influx via a La2+ sensitive L‐type cellular membrane channel. Furthermore, flagellin also acts as a TNF‐α inducer in a time‐ and concentration‐dependent manner when adhered mononuclear cells are treated with flagellin. This ability to induce TNF‐α production was affected by the presence of [Ca2+] in the culture medium. It suggested that B. pseudomallei flagellin is an immuno‐stimulatory molecule, causing an increase in [Ca2+]i and an up‐regulation of TNF‐α, which may play an important role in the inflammation process.

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Salmonella Flagellin-Dependent Proinflammatory Responses Are Localized to the Conserved Amino and Carboxyl Regions of the Protein

Cited by 125 publications

References 27 publications

Identification of a Sequence in Human Toll-like Receptor 5 Required for the Binding of Gram-negative Flagellin

Identification of a Sequence in Human Toll-like Receptor 5 Required for the Binding of Gram-negative Flagellin

Deletion of Flagellin’s Hypervariable Region Abrogates Antibody-Mediated Neutralization and Systemic Activation of TLR5-Dependent Immunity

Immunostimulatory Flagellin from Burkholderia pseudomallei Effects on an Increase in the Intracellular Calcium Concentration and Up‐Regulation of TNF‐α by Mononuclear Cells

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