Sequence, genetic analysis, and expression of Actinobacillus pleuropneumoniae transferrin receptor genes

Abstract

The tbpA and *pi? genes encoding the transferrin receptor proteins T b p l and Tbp2 from a serotype 7 strain of Actinobacillus pleuropneumoniae were cloned, sequenced, and expressed in Escherichia coli. The tbpB gene was preceded by putative promoter and regulatory sequences and was separated from the downstream tbpA gene by a 13 bp intercistronic sequence suggesting that the two genes may be coordinately transcribed. Determination of the nucleotide sequence of this region facilitated PCR amplification of the tbp region from a serotype 1 strain for comparative purposes. The deduced amino acid sequences of the T b p l proteins had regions of homology with Neisseria Lbp and Tbpls and with TonB-dependent outer membrane (OM) receptors of E. coli. The deduced amino acid sequences of the Tbp2 proteins were nearly identical to those presented in previous studies. Upon high-level expression of the tbpA gene, a large proportion of the recombinant T b p l was found in inclusion bodies and could not be aff inity-isolated with immobilized porcine transferrin. Most of the remaining expressed T b p l was present in the OM fraction, was expressed at the surface of E. coli cells, and retained binding activity that was specific for the Globe of porcine transferrin. Although recombinant Tbp2 was found in inclusion bodies during high-level expression, a significant proportion was associated with a novel OM fraction that appeared in sucrose density gradients which was distinct from the OM fraction containing recombinant Tbpl. The recombinant Tbp2 was accessible at the surface yet was unable to bind porcine transferrin. In contrast to previous observations, the binding by recombinant Tbp2 was specific for the Globe of porcine transferrin. These results indicate that the A. pleuropneumoniae transferrin receptor proteins have similar properties to the receptor proteins in Neisseria spp. and Haemophilus influenzae, and that functional studies performed with recombinant receptor proteins need to consider differences in processing and export of these proteins when expressed in heterologous hosts.

Identification of a locus involved in the utilization of iron by Actinobacillus pleuropneumoniae

“…In N. gonorrhoeae and N. meningitidis, two proteins (Tbpl and Tbp2) are responsible for binding transferrin to the cell surface [15]. Similarly, the genes for two transferrin binding proteins (Tbpl and Tbp2) have been cloned and sequenced in A. pleuropneumoniae [6]. In Neisseria species, the iron can be removed from transferrin or lactoferrin to the periplasmic space, and carried by Fbp to transport the iron molecule into the cells [15].…”

“…pleuropneumoniae obtains iron from haem compounds [3] via the production hemolysins [4,5], and membrane-bound transferrin-specific receptors [6]. A. pleuropneumoniae probably binds the iron-loaded transferrin molecule to its surface and then, transports the iron from the transferrin into the cells.…”

Neisseria meningitidis Expressing Transferrin Binding Proteins of Actinobacillus pleuropneumoniae Can Utilize Porcine Transferrin for Growth

“…Oligonucleotide primer sequences are based on the known sequences of the tbp operon in N. meningitidis B16B6 (27) and A. pleuropneumoniae H49 (17) and are listed in Table 1. PCRs were performed according to the manufacturer's instructions, and subsequent cloning steps undertaken with E. coli DH5␣ used standard molecular biology techniques (41).…”

“…The tbp operons from N. meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae (all specific to hTF), Actinobacillus pleuropneumoniae (specific to porcine TF [pTF]), and Pasteurella haemolytica (specific to bovine, sheep, and goat TFs) have been cloned and sequenced (1,17,18,27,33). Recombinant TbpA, recombinant TbpB, and truncated or chimeric TbpB proteins have been expressed and purified from Escherichia coli for binding studies (8,9,14,17,26,29,33,(36)(37)(38). However, the expression of heterologous or chimeric Tbp in members of the Neisseriaceae or Pasteurallaceae families, which would be useful for functional studies, has not been reported.…”

Characterization of a Novel Transferrin Receptor in Bovine Strains of Pasteurella multocida

“…The genes encoding TbpA and TbpB (tbpA and tbpB) are in an operonic arrangement, with tbpB preceding tbpA and putative regulatory and promoter sequences upstream of the tbpB gene (13,14,24). TbpB is a largely surface-exposed lipoprotein, capable of independently binding Tf and participating in the iron acquisition process, but it is not absolutely essential for iron acquisition in vitro (14).…”