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Infection and Immunity, February 2002, p. 732-740, Vol. 70, No. 2
0019-9567/01/$04.00+0     DOI: 70.2.732-740.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Specific Ligand Binding Attributable to Individual Epitopes of Gonococcal Transferrin Binding Protein A

Heather P. Masri and Cynthia Nau Cornelissen*

Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298-0678

Received 2 August 2001/ Returned for modification 9 October 2001/ Accepted 30 October 2001

The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in Escherichia coli, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed.

* Corresponding author. Mailing address: P.O. Box 980678, Richmond, VA 23298-0678. Phone: (804) 827-1754. Fax: (804) 828-9946. E-mail: cncornel{at}hsc.vcu.edu.

Editor: J. T. Barbieri

Infection and Immunity, February 2002, p. 732-740, Vol. 70, No. 2
0019-9567/01/$04.00+0     DOI: 70.2.732-740.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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