Salmonella flagellin-dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein

Tonyia Eaves-Pyles, H. R. Wong, K. Odoms, Richard Pyles

Research output: Contribution to journalArticle

113 Citations (Scopus)

Abstract

Flagellin, the monomeric subunit of flagella, is an inducer of proinflammatory mediators. Bacterial flagellin genes have conserved domains (D1 and D2) at the N terminus and C terminus and a middle hypervariable domain (D3). To identify which domains induced proinflammatory activity, r6-histidine (6HIS)-tagged fusion constructs were generated from the Salmonella dublin (SD) fliC flagellin gene. A full-length r6HIS SD flagellin (6HIS flag) induced IκBα loss poststimulation and NF-κB activation in Caco-2BBe cells and was as potent as native-purified SD flagellin. IFN-γ-primed DLD-1 cells stimulated with 1 μg/ml of 6HIS flag induced high levels of NO (60 ± 0.95 μM) comparable to the combination of IL-1β and IFN-γ (77 ± 1.2) or purified native SD flag (66.3 ± 0.98). Selected rSD flagellin proteins representing the D1, D2, or D3 domains alone or in combination were tested for proinflammatory properties. Fusion proteins representing the D3, amino, or carboxyl regions alone did not induce proinflammatory mediators. The results with a recombinant protein containing the amino D1 and D2 and carboxyl D1 and D2 separated by an Escherichia coli hinge (ND1-2/ECH/CD2) indicated that D1 and D2 were bioactive when coupled to an ECH element to allow protein folding. This chimera, but not the hinge alone, induced IκBα degradation, NF-κB activation, and NO and IL-8 production in two intestinal epithelial cell lines. ND1-2/ECH/CD2-1 also induced high levels of TNF-α (900 pg/ml) in human monocytes comparable to native SD flagellin (991.5 pg/ml) and 6HIS flag (987 pg/ml). The potent proinflammatory activity of flagellin, therefore, resides in the highly conserved N and C D1 and D2 regions.

Original languageEnglish (US)
Pages (from-to)7009-7016
Number of pages8
JournalJournal of Immunology
Volume167
Issue number12
StatePublished - Dec 15 2001

Fingerprint

Flagellin
Salmonella
Proteins
Bacterial Genes
Flagella
Protein Folding
Interleukin-8
Interleukin-1
Recombinant Proteins
Histidine
Monocytes
Epithelial Cells
Escherichia coli
Cell Line

ASJC Scopus subject areas

  • Immunology

Cite this

Salmonella flagellin-dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein. / Eaves-Pyles, Tonyia; Wong, H. R.; Odoms, K.; Pyles, Richard.

In: Journal of Immunology, Vol. 167, No. 12, 15.12.2001, p. 7009-7016.

Research output: Contribution to journalArticle

@article{a01290f8a6e7419aae27626be52edffe,
title = "Salmonella flagellin-dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein",
abstract = "Flagellin, the monomeric subunit of flagella, is an inducer of proinflammatory mediators. Bacterial flagellin genes have conserved domains (D1 and D2) at the N terminus and C terminus and a middle hypervariable domain (D3). To identify which domains induced proinflammatory activity, r6-histidine (6HIS)-tagged fusion constructs were generated from the Salmonella dublin (SD) fliC flagellin gene. A full-length r6HIS SD flagellin (6HIS flag) induced IκBα loss poststimulation and NF-κB activation in Caco-2BBe cells and was as potent as native-purified SD flagellin. IFN-γ-primed DLD-1 cells stimulated with 1 μg/ml of 6HIS flag induced high levels of NO (60 ± 0.95 μM) comparable to the combination of IL-1β and IFN-γ (77 ± 1.2) or purified native SD flag (66.3 ± 0.98). Selected rSD flagellin proteins representing the D1, D2, or D3 domains alone or in combination were tested for proinflammatory properties. Fusion proteins representing the D3, amino, or carboxyl regions alone did not induce proinflammatory mediators. The results with a recombinant protein containing the amino D1 and D2 and carboxyl D1 and D2 separated by an Escherichia coli hinge (ND1-2/ECH/CD2) indicated that D1 and D2 were bioactive when coupled to an ECH element to allow protein folding. This chimera, but not the hinge alone, induced IκBα degradation, NF-κB activation, and NO and IL-8 production in two intestinal epithelial cell lines. ND1-2/ECH/CD2-1 also induced high levels of TNF-α (900 pg/ml) in human monocytes comparable to native SD flagellin (991.5 pg/ml) and 6HIS flag (987 pg/ml). The potent proinflammatory activity of flagellin, therefore, resides in the highly conserved N and C D1 and D2 regions.",
author = "Tonyia Eaves-Pyles and Wong, {H. R.} and K. Odoms and Richard Pyles",
year = "2001",
month = "12",
day = "15",
language = "English (US)",
volume = "167",
pages = "7009--7016",
journal = "Journal of Immunology",
issn = "0022-1767",
publisher = "American Association of Immunologists",
number = "12",

}

TY - JOUR

T1 - Salmonella flagellin-dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein

AU - Eaves-Pyles, Tonyia

AU - Wong, H. R.

AU - Odoms, K.

AU - Pyles, Richard

PY - 2001/12/15

Y1 - 2001/12/15

N2 - Flagellin, the monomeric subunit of flagella, is an inducer of proinflammatory mediators. Bacterial flagellin genes have conserved domains (D1 and D2) at the N terminus and C terminus and a middle hypervariable domain (D3). To identify which domains induced proinflammatory activity, r6-histidine (6HIS)-tagged fusion constructs were generated from the Salmonella dublin (SD) fliC flagellin gene. A full-length r6HIS SD flagellin (6HIS flag) induced IκBα loss poststimulation and NF-κB activation in Caco-2BBe cells and was as potent as native-purified SD flagellin. IFN-γ-primed DLD-1 cells stimulated with 1 μg/ml of 6HIS flag induced high levels of NO (60 ± 0.95 μM) comparable to the combination of IL-1β and IFN-γ (77 ± 1.2) or purified native SD flag (66.3 ± 0.98). Selected rSD flagellin proteins representing the D1, D2, or D3 domains alone or in combination were tested for proinflammatory properties. Fusion proteins representing the D3, amino, or carboxyl regions alone did not induce proinflammatory mediators. The results with a recombinant protein containing the amino D1 and D2 and carboxyl D1 and D2 separated by an Escherichia coli hinge (ND1-2/ECH/CD2) indicated that D1 and D2 were bioactive when coupled to an ECH element to allow protein folding. This chimera, but not the hinge alone, induced IκBα degradation, NF-κB activation, and NO and IL-8 production in two intestinal epithelial cell lines. ND1-2/ECH/CD2-1 also induced high levels of TNF-α (900 pg/ml) in human monocytes comparable to native SD flagellin (991.5 pg/ml) and 6HIS flag (987 pg/ml). The potent proinflammatory activity of flagellin, therefore, resides in the highly conserved N and C D1 and D2 regions.

AB - Flagellin, the monomeric subunit of flagella, is an inducer of proinflammatory mediators. Bacterial flagellin genes have conserved domains (D1 and D2) at the N terminus and C terminus and a middle hypervariable domain (D3). To identify which domains induced proinflammatory activity, r6-histidine (6HIS)-tagged fusion constructs were generated from the Salmonella dublin (SD) fliC flagellin gene. A full-length r6HIS SD flagellin (6HIS flag) induced IκBα loss poststimulation and NF-κB activation in Caco-2BBe cells and was as potent as native-purified SD flagellin. IFN-γ-primed DLD-1 cells stimulated with 1 μg/ml of 6HIS flag induced high levels of NO (60 ± 0.95 μM) comparable to the combination of IL-1β and IFN-γ (77 ± 1.2) or purified native SD flag (66.3 ± 0.98). Selected rSD flagellin proteins representing the D1, D2, or D3 domains alone or in combination were tested for proinflammatory properties. Fusion proteins representing the D3, amino, or carboxyl regions alone did not induce proinflammatory mediators. The results with a recombinant protein containing the amino D1 and D2 and carboxyl D1 and D2 separated by an Escherichia coli hinge (ND1-2/ECH/CD2) indicated that D1 and D2 were bioactive when coupled to an ECH element to allow protein folding. This chimera, but not the hinge alone, induced IκBα degradation, NF-κB activation, and NO and IL-8 production in two intestinal epithelial cell lines. ND1-2/ECH/CD2-1 also induced high levels of TNF-α (900 pg/ml) in human monocytes comparable to native SD flagellin (991.5 pg/ml) and 6HIS flag (987 pg/ml). The potent proinflammatory activity of flagellin, therefore, resides in the highly conserved N and C D1 and D2 regions.

UR - http://www.scopus.com/inward/record.url?scp=0035892911&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035892911&partnerID=8YFLogxK

M3 - Article

VL - 167

SP - 7009

EP - 7016

JO - Journal of Immunology

JF - Journal of Immunology

SN - 0022-1767

IS - 12

ER -