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Infection and Immunity, December 2008, p. 5524-5534, Vol. 76, No. 12
0019-9567/08/$08.00+0     doi:10.1128/IAI.00843-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vibrio cholerae Flagellins Induce Toll-Like Receptor 5-Mediated Interleukin-8 Production through Mitogen-Activated Protein Kinase and NF-{kappa}B Activation{triangledown}

Lisa M. Harrison, Prasad Rallabhandi, Jane Michalski, Xin Zhou, Susan R. Steyert, Stefanie N. Vogel, and James B. Kaper*

Department of Microbiology and Immunology, University of Maryland Baltimore School of Medicine, Baltimore, Maryland 21201

Received 8 July 2008/ Returned for modification 3 September 2008/ Accepted 17 September 2008

Vaccine reactogenicity has complicated the development of safe and effective live, oral cholera vaccines. {Delta}ctx Vibrio cholerae mutants have been shown to induce inflammatory diarrhea in volunteers and interleukin-8 (IL-8) production in cultured intestinal epithelial cells. Bacterial flagellins are known to induce IL-8 production through Toll-like receptor 5 (TLR5). Since the V. cholerae genome encodes five distinct flagellin proteins, FlaA to FlaE, with homology to conserved TLR5 recognition regions of Salmonella FliC, we hypothesized that V. cholerae flagellins may contribute to IL-8 induction through TLR5 and mitogen-activated protein kinase (MAPK) signaling. Each purified recombinant V. cholerae flagellin induced IL-8 production in T84 intestinal epithelial cells and also induced nuclear factor kappa B (NF-{kappa}B) activation in HEK293T/TLR5 transfectants, which was blocked by cotransfection with a TLR5 dominant-negative construct, demonstrating TLR5 specificity. Supernatants derived from {Delta}flaAC and {Delta}flaEDB mutants induced IL-8 production in HT-29 intestinal epithelial cells and in HEK293T cells overexpressing TLR5, whereas {Delta}flaABCDE supernatants induced significantly less IL-8 production, demonstrating the contribution of multiple flagellins in IL-8 induction. NF-{kappa}B activation by {Delta}flaABCDE supernatants was partially restored by flaA or flaAC complementation. Western analysis confirmed the presence of V. cholerae flagellins in culture supernatants. Purified recombinant V. cholerae FlaA activated the MAPKs p38, c-jun N-terminal kinase (JNK), and extracellular regulated kinase (ERK) in T84 cells. FlaA-induced IL-8 production in T84 cells was inhibited by the p38 inhibitor in combination with either the JNK or ERK inhibitors. Collectively, these data suggest that V. cholerae flagellins are present in culture supernatants and can induce TLR5- and MAPK-dependent IL-8 secretion in host cells.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, 660 W. Redwood St., Suite 324, Baltimore, MD 21201. Phone: (410) 706-3004. Fax: (410) 706-0182. E-mail: jkaper{at}medicine.umaryland.edu

{triangledown} Published ahead of print on 22 September 2008.

Editor: V. J. DiRita

Infection and Immunity, December 2008, p. 5524-5534, Vol. 76, No. 12
0019-9567/08/$08.00+0     doi:10.1128/IAI.00843-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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