MAPPING OF FUNCTIONAL REGIONS ON THE TRANSFERRIN-BINDING PROTEIN (TFBA) OF ACTINOBACILLUS-PLEUROPNEUMONIAE

Actinobacillus pleuropneumoniae can use porcine transferrin as the sol e source of iron. Two proteins with molecular masses of approximately 60 kDa (TfbA) and 110 kDa have been shown to specifically bind porcine transferrin; from the TfbA protein, three isoforms from A. pleuropneu moniae serotypes 1, 5, and 7 have been identified and characterized by nucleotide sequence analysis. Here we defined the transferrin-binding region(s) of the TfbA protein of A. pleuropneumoniae serotype 7 by Tn phoA mutagenesis, random mutagenesis, and peptide spot synthesis. The amino-terminal half of the TfbA molecule, which has only 36% amino aci d sequence identity among the three isoforms, was shown to be responsi ble for transferrin binding by TnphoA mutagenesis. This result was con firmed by analysis of six random mutants with decreased transferrin bi nding affinity. The subsequent analysis of overlapping 16-mer peptides comprising the amino-terminal half of the TfbA molecule revealed thre e domains of 13 or 14 amino acids in length with transferrin-binding a ctivity. They overlapped, or were very close to, point mutations decre asing transferrin-binding ability. The first and third domains were un ique to the TfbA protein of A. pleuropneumoniae serotype 7. In contras t, the sequence of the second domain was present in almost identical f orms (12 of 14 residues) in the TfbA proteins of A. pleuropneumoniae s erotypes 1 and 5; in addition, a sequence consisting of functionally h omologous amino acids was present in the otherwise completely distinct small transferrin-binding proteins of Neisseria gonorrhoeae (TbpB), N . meningitidis (Tbp2), and Haemophilus influenzae (Tbp2).