SEQUENCE, GENETIC-ANALYSIS, AND EXPRESSION OF ACTINOBACILLUS-PLEUROPNEUMONIAE TRANSFERRIN RECEPTOR GENES

The tbpA and tbpB genes encoding the transferrin receptor proteins Tbp 1 and Tbp2 from a serotype 7 strain of Actinobacillus pleuropneumoniae were cloned, sequenced, and expressed in Escherichia coli. The tbpB g ene was preceded by putative promoter and regulatory sequences and was separated from the downstream tbpA gene by a 13 bp intercistronic seq uence suggesting that the two genes may be coordinately transcribed. D etermination of the nucleotide sequence of this region facilitated PCR amplification of the tbp region from a serotype 1 strain for comparat ive purposes. The deduced amino acid sequences of the Tbp1 proteins ha d regions of homology with Neisseria Lbp and Tbp1s and with TonB-depen dent outer membrane (OM) receptors of E. coli. The deduced amino acid sequences of the Tbp2 proteins were nearly identical to those presente d in previous studies. Upon high-level expression of the tbpA gene, a large proportion of the recombinant Tbp1 was found in inclusion bodies and could not be affinity-isolated with immobilized porcine transferr in. Most of the remaining expressed Tbp1 was present in the OM fractio n, was expressed at the surface of E. coli cells, and retained binding activity that was specific for the C-lobe of porcine transferrin. Alt hough recombinant Tbp2 was found in inclusion bodies during high-level expression, a significant proportion was associated with a novel OM f raction that appeared in sucrose density gradients which was distinct from the OM fraction containing recombinant Tbp1. The recombinant Tbp2 was accessible at the surface yet was unable to bind porcine transfer rin. In contrast to previous observations, the binding by recombinant Tbp2 was specific for the C-lobe of porcine transferrin. These results indicate that the A. pleuropneumoniae transferrin receptor proteins h ave similar properties to the receptor proteins in Neisseria spp. and Haemophilus influenzae, and that functional studies performed with rec ombinant receptor proteins need to consider differences in processing and export of these proteins when expressed in heterologous hosts.