Isolation and identification of a putative porcine transferrin receptor from Actinobacillus pleuropneumoniae biotype 1

Abstract

~ ~ ~~~Each of two affinity isolation methods, the first based on biotinylated porcine transferrin plus streptavidinagarose, and the second on Sepharose-coupled porcine transferrin, followed by SDS-PAGE, allowed the isolation and identification of two potential porcine-transferrin-binding polypeptides ( -64 kDa and 99 kDa) from total membranes of Actinobaciflus pfeuropneumoniae 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. While these results suggest that the 99 kDa polypeptide represents the porcine transferrin receptor of A. pfeuropneumoniae, and that the 64 kDa polypeptide represents an associated protein serving an accessory role, other interpretations are also possible.

Bacterial transferrin receptors ? structure, function and contribution to virulence

“…Two iron-repressible porcine transferrin-binding proteins of 56 and 105 kDa, respectively, have been isolated from A. pleuropneumoniae serotypes l, 2, and 7, of which only the 56-kDa protein could be renatured to bind porcine transferrin after SDS-PAGE and Western blotting [32]. Similar results were obtained by Ricard et al [78], who reported the purification oftransferrin binding proteins of 64 and 99 kDa from A. pleuropneumoniae.…”

“…Interestingly, however, strain Eagan appears to be an exception possessing TBPs of 107 kDa and 76 kDa, respectively, although the latter may represent a partially unfolded 90-kDa TBP2 [46]. The transferrin binding proteins of A.pleuropneumoniae [32,78] and 1t. somnus [70] have also been isolated using similar affinity-isolation methods but employing porcine and bovine transferrins respectively.…”

Acquisition of haemoglobin-bound iron by Histophilus somni

“…A polypeptide with an estimated molecular mass of -120 kDa was detected in total and outer membranes from all three strains but only if the membranes were from organisms grown under the conditions that promoted the expression ofHb binding activity. Unfortunately, the -120-kDa polypeptides could not be affmity-iso1ated using any of the methods described by Ricard et al (1991), Elkins (1995) or Jin et al (1996), with or without modifications.…”

“…The methods of Ricard et al (1991) and Jin et al (1996), using biotinylated bovine Hb as the binding ligand, were also tried but without success. The effectiveness of the solubilizing solutions was also questioned.…”

Haemophilus somnus Possesses Two Systems for Acquisition of Transferrin-Bound Iron

“…Tf-binding polypeptides were isolated from total membranes of H. somnus strain 649 by using the affinity procedure developed by Schryvers and Morris (22), as modified by Ricard et al (20). The membranes used in these experiments were from organisms grown under iron-restricted conditions in the presence of Tf, since such growth conditions were the only ones that yielded organisms exhibiting significant Tf-binding activity.…”