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Infection and Immunity, December 2009, p. 5437-5448, Vol. 77, No. 12
0019-9567/09/$08.00+0     doi:10.1128/IAI.00666-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Molecular, Antigenic, and Functional Characteristics of Ferric Enterobactin Receptor CfrA in Campylobacter jejuni {triangledown}

Ximin Zeng,{dagger} Fuzhou Xu,{dagger} and Jun Lin*

Department of Animal Science, The University of Tennessee, 2505 River Drive, Knoxville, Tennessee 37996

Received 10 June 2009/ Returned for modification 15 July 2009/ Accepted 29 August 2009

The ferric enterobactin receptor CfrA not only is responsible for high-affinity iron acquisition in Campylobacter jejuni but also is essential for C. jejuni colonization in animal intestines. In this study, we determined the feasibility of targeting the iron-regulated outer membrane protein CfrA for immune protection against Campylobacter colonization. Alignment of complete CfrA sequences from 15 Campylobacter isolates showed that the levels of amino acid identity for CfrA range from 89% to 98%. Immunoblotting analysis using CfrA-specific antibodies demonstrated that CfrA was dramatically induced under iron-restricted conditions and was widespread and produced in 32 Campylobacter primary strains from various sources and from geographically diverse areas. The immunoblotting survey results were highly correlated with the results of an enterobactin growth promotion assay and a PCR analysis using cfrA-specific primers. Inactivation of the cfrA gene also impaired norepinephrine-mediated growth promotion, suggesting that CfrA is required for C. jejuni to sense intestinal stress hormones during colonization. Complementation of the cfrA mutant with a wild-type cfrA allele in trans fully restored the production and function of CfrA. A growth assay using purified anti-CfrA immunoglobulin G demonstrated that specific CfrA antibodies could block the function of CfrA, which diminished ferric enterobactin-mediated growth promotion under iron-restricted conditions. The inhibitory effect of CfrA antibodies was dose dependent. Immunoblotting analysis also indicated that CfrA was expressed and immunogenic in chickens experimentally infected with C. jejuni. Amino acid substitution mutagenesis demonstrated that R327, a basic amino acid that is highly conserved in CfrA, plays a critical role in ferric enterobactin acquisition in C. jejuni. Together, these findings strongly suggest that CfrA is a promising vaccine candidate for preventing and controlling Campylobacter infection in humans and animal reservoirs.

* Corresponding author. Mailing address: Department of Animal Science, The University of Tennessee, 2640 Morgan Circle Drive, Knoxville, TN 37996-4574. Phone: (865) 974-5598. Fax: (865) 974-7297. E-mail: jlin6{at}utk.edu

{triangledown} Published ahead of print on 8 September 2009.

Editor: A. J. Bäumler

{dagger} X.Z. and F.X. contributed equally to this work.

Infection and Immunity, December 2009, p. 5437-5448, Vol. 77, No. 12
0019-9567/09/$08.00+0     doi:10.1128/IAI.00666-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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