No description available.

Seven strains of Actinobacillus suis (ATCC 15557, B49, C84, H89-1173, H91-0380, SO4 and VSB 3714) were investigated with respect to iron acquisition from animal transferrins (Tfs) and haemoglobins (Hbs). Growth assays with porcine, bovine and human Tfs and Hbs revealed that all seven strains could use porcine (but not human or bovine) Tf and all three Hbs as iron sources. In solid phase binding assays, membranes derived from all strains exhibited strong binding of porcine Tf and each of the Hbs. Competition binding assays indicated that all three Hbs were bound by the same receptor(s). Affinity procedures allowed the isolation and identification of iron repressible Tf-binding (~100 kDa and ~63 kDa) and Hb-binding (~105 kDa) polypeptides from all strains. Nucleotide sequence analyses revealed that A. suis strains SO4 and C84 possess genes that encode homologues of the Actinobacillus pleuropneumoniae Tf-binding proteins, TbpA and TbpB, and Hb-binding protein, HgbA. In both strains, tbpB was located immediately upstream of tbpA and was shown to be preceded by tonB, exbB and exbD homologues; hgbA was shown to be preceded by a hugZ homologue. Putative promoter and Fur box sequences were located upstream of tonB and hugZ and RT-PCR revealed that the genes in each of these clusters (tonB-exbB-exbD-tbpB-tbpA; hugZ-hgbA) are co-transcribed and iron-repressible. The molecular masses of the predicted mature TbpA, TbpB and HgbA proteins were calculated to be 104.3, 63.4 and 105.0 kDa, respectively, suggesting that the affinity-isolated, ~100 kDa and ~63 kDa Tf-binding polypeptides represent TbpA and TbpB, respectively, and that the ~105 kDa Hb-binding polypeptide represents HgbA. TbpB of A. suis was expressed in Escherichia coli and the recombinant TbpB (rTbpB) was identified by immunoblotting using swine sera raised against recombinant TbpB (A. pleuropneumoniae). It is envisaged that the acquisition of Tf- and Hb-bound iron by A. suis involves mechan

Each of two affinity isolation methods, the first based on biotinylated porcine transferrin plus streptavidin-agarose, and the second on Sepharose-coupled porcine transferrin, followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), allowed the isolation and identification of two potential porcine-transferrin-binding polypeptides (∼64 kDa and 99 kDa) from total membranes of Actinobacillus pleuropneumoniae grown under iron-restricted conditions. Both polypeptides were iron-repressible and were identified as potential receptor candidates as they were not isolated when biotinylated human transferrin was used instead of biotinylated porcine transferrin. The 64 kDa polypeptide was the more easily removed from Sepharose-coupled porcine transferrin and only the 99 kDa polypeptide appeared to be an outer membrane protein. These results suggest that the 99 kDa polypeptide represents the porcine transferrin receptor of A. pleuropneumoniae, and that the 64 kDa polypeptide represents an associated protein serving an accessory role. Fe+3 uptake studies using plate assays and attempted isolation of a putative lactoferrin receptor using biotinylated porcine lactoferrin plus streptavidin-agarose, followed by SDS-PAGE, showed that A. pleuropneumoniae lacks a mechanism for the use of porcine lactoferrin as an iron source.

No description available.