Genetic and functional analysis of Haemophilus somnus high molecular weight-immunoglobulin binding proteins.
Haemophilus somnus immunoglobulin binding proteins (IgBPs) are virulence associated but only one (p76) has been genetically defined. We determined the nucleotide sequence of the 5'-flanking region of the p76 gene. This region had been identified as the coding region for a series of high molecular weight (HMW)-IgBPs. Analysis of the nucleotide sequence indicated the gene (immunoglobulin binding protein A, ibpA) encoding the HMW and p76 IgBPs comprised a single open reading frame of 12,285 base pairs (bp). The ibpA gene is flanked by an upstream ORF of 1758bp, designated ibpB. The predicted amino acid sequences of these two genes demonstrate similarity to virulence exoproteins and their transporter proteins that comprise a two-partner secretion pathway in various Gram-negative bacteria. Motifs associated with binding to mammalian cells were also identified within the sequence. Competitive inhibition studies implicated a putative heparin-binding domain in adherence to bovine endothelial cells. Expression plasmids for glutathione S-transferase (GST)-fused recombinant fragments covered amino acid residues 972-3201. IgG2 Fc binding studies identified fragment 972-1515 (GST-IbpA3) as an Fc binding peptide. This peptide and GST-IbpA5 (aa 2071-2730) reacted strongly with convalescent phase serum. In a small preliminary study, calves immunized with the purified GST-IbpA3 peptide were protected against an intrabronchial H. somnus challenge.
Tagawa
Yuichi
Y
Department of Pathology, University of California, San Diego Medical Center, 200 West Arbor Drive, San Diego, CA 92103-8416, USA. ytagawa@affrc.go.jp
Sanders
Jerry D
JD
Uchida
Ikuo
I
Bastida-Corcuera
Felix D
FD
Kawashima
Kenji
K
Corbeil
Lynette B
LB
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D004730:Endothelium, Vascular / Q000382:microbiology; D006192:Haemophilus Infections / Q000276:immunology / Q000473:pathology / Q000517:prevention & control; D044138:Haemophilus somnus / Q000235:genetics / Q000502:physiology*; D006493:Heparin / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D007136:Immunoglobulins / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011485:Protein Binding; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D037521:Virulence Factors / Q000235:genetics
D001426:Bacterial Proteins; D007141:Immunoglobulin Fc Fragments; D007136:Immunoglobulins; D011994:Recombinant Proteins; D037521:Virulence Factors; D006493:Heparin