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

Abstract

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.

Identification and Characterization of a New Ferric Enterobactin Receptor, CfrB, in Campylobacter

“…Interestingly, in this study one C. jejuni strain (JL11), which does not have a gene highly homologous to cfrA, could efficiently utilize FeEnt as a sole iron source for growth (40). An early study also showed that an isogenic cfrA mutant of a human C. coli strain was still fully capable of utilizing FeEnt as a sole iron source for growth (15).…”

“…Among the 77 strains used for a survey of prevalence and function, 32 isolates were C. jejuni while 45 isolates were C. coli. For the C. jejuni strains, the majority of them (30 out of 32) were described and used in our previous CfrA study (40), and 2 new human isolates were used in this study. The C. coli strains were isolated from human (5 isolates), bovine (10 isolates), swine (18 isolates), chicken (11 isolates), and turkey (1 isolate) samples.…”

“…An isogenic cfrB mutant of C. coli JL170 was constructed by using the same insertional mutagenesis strategy as described previously (40). Briefly, based on the published genome sequence of C. coli RM2228 (13), primers CCfrBF and CCfrBR (Table 2) were designed for successful amplification of a 2.7-kb cfrB fragment from C. coli JL170.…”

“…A transformant with a plasmid construct bearing the intact cfrB gene (named pCfrB) was identified (JL463). The pCfrB plasmid from strain JL463 was then transferred to JL324 (Table 1) by triparental conjugation (40), creating the construct JL465 (Table 1).…”

“…The FeEnt receptor CfrA is induced under iron-restricted conditions and plays a critical role in iron acquisition and in vivo colonization by C. jejuni (27). A recent report (40) provides further molecular, antigenic, and functional evidence suggesting that CfrA is a promising subunit vaccine for preventing and controlling C. jejuni infection in humans and animal reservoirs.…”