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Journal of Bacteriology, February 2004, p. 850-857, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.850-857.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Transferrin-Binding Protein B of Neisseria meningitidis: Sequence-Based Identification of the Transferrin-Binding Site Confirmed by Site-Directed Mutagenesis

Geneviève Renauld-Mongénie,^1* Laurence Lins,² Tino Krell,¹ Laure Laffly,¹ Michèle Mignon,¹ Monique Dupuy,¹ Rose-May Delrue,^1, Françoise Guinet-Morlot,¹ Robert Brasseur,² and Ling Lissolo¹

Aventis Pasteur, 69280 Marcy l'Etoile, France,¹ Centre de Biophysique Moleculaire Numerique, Faculté des Sciences Agronomiques de Gembloux, Gembloux, Belgium²

Received 29 August 2003/ Accepted 24 October 2003

A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.

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* Corresponding author. Mailing address: Aventis Pasteur, 1541 Ave. Marcel Mérieux, 69280 Marcy l'Etoile, France. Phone: (33) 4 37 37 35 05. Fax: (33) 4 37 37 36 39. E-mail: genevieve.renauld{at}aventis.com.

Present address: Unité de Recherche en Biologie Moléculaire, FUNDP, Namur, Belgium.

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Journal of Bacteriology, February 2004, p. 850-857, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.850-857.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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