Two Nonadjacent Regions in Enteroaggregative Escherichia coli Flagellin Are Required for Activation of Toll-like Receptor 5

Donnelly, Meghan; Steiner, Theodore S.

doi:10.1074/jbc.m206851200

Cited by 135 publications

(101 citation statements)

References 24 publications

(35 reference statements)

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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%

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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%

“…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%

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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“…6). 110 Further exploration of the correlates of the primary sequence of flagellin and TLR5 agonism have now defined two regions: a 14-residue N-motif peptide (AA 95-108) and a 9-residue C-motif peptide (AA 441-449), deletions or mutagenesis of which result in abrogation of TLR5 signaling. 111 Flagellin has been demonstrated to induce maturation and chemokine production in human dendritic cells, 112 resulting in stimulation of T H 1 responses via IL-12p70 production, which depends on the phosphorylation of p38 and c-Jun N-terminal kinase 1/2 (JNK).…”

Section: Tlr5 Agonistic Flagellinmentioning

confidence: 99%

“…109 Random and site-directed mutagenesis of flagellin from enteroaggregative E. coli identified two regions in the conserved D1 domain that are required for interleukin-8 release and TLR5 activation in Caco-2 cells. 110 The transposon linker insertions which eliminate inflammatory activity (2H3, E8, F11, C5) are located near the midpoint of the α-helix formed by the C-terminal conserved domain of the protein (Fig. 6).…”

Section: Tlr5 Agonistic Flagellinmentioning

confidence: 99%

Potential adjuvantic properties of innate immune stimuli

et al. 2009

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Just as the prescient comment by Gaston Ramon was relegated to the last footnote of his 1926 paper, 1 so has research on the mechanisms of action of adjuvants, until recently, languished as parenthetical annotations and addenda in the archives of immunology and vaccine development. Ramon defined immunological adjuvants as "substances used in combination with a specific antigen that produced a more robust immune response than the antigen alone." Interestingly enough, he was referring to his empirical findings that the addition of bread crumbs, tapioca, saponin and 'starch oil' to antigenic preparations greatly enhanced antibody responses to diphtheria or tetanus. 2 A year later, the adjuvanticity of aluminum salts (primarily phosphate and hydroxide) was discovered by Glenny and coworkers. 3 In the 83 years that have elapsed, the repertoire of investigational adjuvants has grown to encompass a very wide range of materials, 4 but aluminum salt-based mineral salts (generically, and incorrectly, termed "alum") have remained the only adjuvants currently approved by the FDA. Aluminum salts have enjoyed a good safety record, but they are weak adjuvants for antibody induction and induce a T helper-2 (T H 2)-skewed, rather than a T helper-1 (T H 1) response. 5,6 Furthermore, not only are aluminum salts ineffective at inducing cytotoxic T lymphocyte (CTL) or mucosal IgA antibody responses, but also have a propensity to induce IgE responses, which have been associated with allergic reactions in some subjects. 5,6 Very recent reports implicate the Nalp3 inflammasome, a component of the innate immune response, as the effector limb of alum-associated adjuvanticity. [7][8][9] In 1962, Dresser observed that injection of purified soluble proteins not only failed to stimulate an immune response, but tolerized animals unless a bacterial extract was admixed with the protein immunogen. 10 This led him to redefine adjuvanticity as "a property of a substance which can act as a physiological switch, directing at least some immunologically competent cells to respond by making antibody rather than by becoming immunologically paralyzed by the antigen," 11 confirming Johnson's earlier observations that lipopolysaccharide (LPS) from Gram-negative bacteria exerted potent adjuvant properties, 12 and perhaps paved the way for the subsequent discovery of the wide range of microorganism-derived adjuvants. 13 TLRs are pattern recognition receptors present on diverse cell types that recognize specific molecular patterns present in molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs). 14,15 There are 10 TLRs in the human genome;

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“…8 Flagellin contains two highly conserved N/C domains (D0 and D1) and one central hypervariable domain (D2/D3). [9][10][11] The hypervariable domain is vastly diverse in size and amino-acid composition among bacterial strains and species. 4 The conserved D0 and D1 domains are required for the immune activity of flagellin as a pathogen-associated molecular pattern.…”

Section: Introductionmentioning

confidence: 99%

Over-activation of TLR5 signaling by high-dose flagellin induces liver injury in mice

et al. 2014

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Flagellin is a potent activator of a broad range of cell types that are involved in innate and adaptive immunity. Therefore, it is a good adjuvant candidate for vaccines, and it might function as a biological protectant against both major acute radiation syndrome during cancer radiotherapy and a mitigator of radiation emergencies. However, accumulating evidence has implicated flagellin in the occurrence of some inflammatory diseases, such as acute lung inflammation, cardiovascular collapse and inflammatory bowel disease. The aim of this study was to elucidate whether only flagellin-TLR5 signaling activation plays a role in the pathophysiology of liver or whether some other flagellin activity also contributes to liver injury either via bacterial infections or during clinical applications. Recombinant flagellin proteins with or without TLR5-stimulating activity were used to evaluate the role of flagellin-TLR5 signaling in liver injury in wild-type and TLR5 KO mice. Gross lesions and large areas of hepatocellular necrosis were observed in liver tissue 12 h after the intraperitoneal administration of 100 or 200 mg flagellin (FliC) in a dose-and time-dependent manner in wild-type mice, but not in TLR5 KO mice. Deletion of the N-terminal or TLR5 binding domain of flagellin inhibited flagellin-induced inflammatory responses and the subsequent acute liver function abnormality and damage. These data confirmed that flagellin is an essential determinant of liver injury and demonstrated that the over-activation of TLR5 signaling by high-dose flagellin caused acute inflammatory responses, neutrophil accumulation and oxidative stress in the liver, which contributes to the progression and severity of flagellin-induced liver injury.

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Two Nonadjacent Regions in Enteroaggregative Escherichia coli Flagellin Are Required for Activation of Toll-like Receptor 5

Cited by 135 publications

References 24 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

Potential adjuvantic properties of innate immune stimuli

Over-activation of TLR5 signaling by high-dose flagellin induces liver injury in mice

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