Mapping of functional regions on the transferrin-binding protein (TfbA) of Actinobacillus pleuropneumoniae

Abstract

Actinobacillus pleuropneumoniae can use porcine transferrin as the sole 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. pleuropneumoniae 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 TnphoA mutagenesis, random mutagenesis, and peptide spot synthesis. The amino-terminal half of the TfbA molecule, which has only 36% amino acid sequence identity among the three isoforms, was shown to be responsible for transferrin binding by TnphoA mutagenesis. This result was confirmed by analysis of six random mutants with decreased transferrin binding affinity. The subsequent analysis of overlapping 16-mer peptides comprising the amino-terminal half of the TfbA molecule revealed three domains of 13 or 14 amino acids in length with transferrin-binding activity. They overlapped, or were very close to, point mutations decreasing transferrin-binding ability. The first and third domains were unique to the TfbA protein of A. pleuropneumoniae serotype 7. In contrast, the sequence of the second domain was present in almost identical forms (12 of 14 residues) in the TfbA proteins of A. pleuropneumoniae serotypes 1 and 5; in addition, a sequence consisting of functionally homologous amino acids was present in the otherwise completely distinct small transferrin-binding proteins of Neisseria gonorrhoeae (TbpB), N. meningitidis (Tbp2), and Haemophilus influenzae (Tbp2).

Identification of sequences in human transferrin that bind to the bacterial receptor protein, transferrin‐binding protein B

“…It is interesting to note that this binding was demonstrated with a very sensitive reporter system and was not compared with the binding of host (human) Tf. The potential for loss of host-speci®c binding under certain conditions is not totally unexpected, as there are a number of TbpB peptide regions involved in binding Tf (Strutzberg et al, 1995;Renauld-Monge  nie et al, 1997), and several of these peptides have been Q 1999 Blackwell Science Ltd, Molecular Microbiology, 32, 111±121 Fig. 4.…”

“…reported not to maintain the strict host speci®city of binding (Strutzberg et al, 1995). Thus, initial binding by segments of TbpB recognizing sequences/structures unique to hTf may be followed by interactions involving regions of hTf that are common to other Tfs.…”

Characterization of the porcinetransferringene (TF ) and its association with disease severity following an experimentalActinobacillus pleuropneumoniaeinfection

“…The pathogen specifically binds porcine TF , and the ability to use TF-bound iron is required for virulence (Baltes et al 2002). Further, the TF receptor is highly specific , and it has been shown that the A. pleuropneumoniae serotypes 1 and 5, being most prevalent in North America, have distinct TF receptor alleles from A. pleuropneumoniae serotype 2, being most prevalent in Northern Europe (Gerlach et al 1992;Strutzberg et al 1995).…”

Infección por Actinobacillus pleuropneumoniae en un modelo murino = Actinobacillus pleuropneumoniae infection in a murine model

“…La proteína TbpB es una lipoproteína que se encuentra expuesta en la superficie de la bacteria, siendo posible la existencia de dominios transmembrana que le permitirían extenderse hacia la membrana interna (Gerlach et al, 1992), mientras que el resto se proyectaría hacia el exterior. Los anticuerpos obtenidos a partir de animales convalecientes de la infección no reconocen el extremo N-terminal de la proteína (necesaria para que se una de forma estable a la transferrina), lo que pone de manifiesto una cierta variabilidad entre sus isoformas (Strutzberg, 1995); sin embargo, la región C-terminal está muy conservada (Gerlach et al, 1992).…”

“…Posteriormente, se ensayaron conjugados de Apx con polisacáridos capsulares. que la región de la proteína principalmente reconocida por anticuerpos es la Cterminal, que también es la más conservada entre los diferentes serotipos(Strutzberg et al, 1995). Se ha descrito un preparado inmunógeno basado en el sobrenadante de un cultivo obtenido en condiciones de restricción de hierro, compuesto básicamente por hemolisinas y proteínas no integrales de membrana externa, entre ellas la TbpB.…”