Cloning and sequencing of the gene encoding a 31-kilodalton antigen of Haemophilus somnus

Abstract

Immunoblots using bovine antibody against Haemophilus somnus as the primary antibody consistently identified 31-, 40- and 78-kDa proteins in Sarkosyl-insoluble extracts of H. somnus. A genomic library of H. somnus 8025 DNA was constructed in plasmid pUC19, and 45 recombinants expressed proteins which were recognized by bovine antiserum in Western blots (immunoblots). Ten of the recombinants expressing a 31-kDa protein caused the lysis of bovine erythrocytes. Restriction endonuclease mapping indicated that the hemolytic recombinants shared an approximately 1.7-kb BglII fragment. Southern blot analysis using the BglII fragment as a probe revealed homology among the recombinants and the presence of an identically sized BglII fragment in the chromosome of all H. somnus isolates tested. Sequence analysis indicated the presence of an 822-bp open reading frame within the 1.7-kb BglII fragment. Deletion of this open reading frame resulted in the loss of hemolytic activity and protein expression in recombinant Escherichia coli, suggesting the possible role of the 31-kDa protein as a hemolysin. An amino acid sequence deduced from the DNA sequence shared homology with outer membrane protein A of E. coli, Salmonella typhimurium, and Shigella dysenteriae, with P6 of Haemophilus influenzae, and with PIII of Neisseria gonorrhoeae. An amino acid analysis of the recombinant 31-kDa protein agreed with the amino acid composition deduced from the DNA sequence.

Characterization of a fourth lipoprotein fromPasteurella haemolyticaA1 and its homology to the OmpA family of outer membrane proteins

“…A homology search comparing Plp4 with sequences in GenBank showed significant homologies with several prokaryotic outer membrane proteins and lipoproteins. The greatest similarity was detected with a 31‐kDa ‘hemolytic’ antigen of H. somnus [10]. The amino acids of Plp4 and the H. somnus antigen are 63% identical and 13.9% similar for a total of 76.9% homology over 273 amino acids.…”

“…We have identified at least four other homologous antigen genes within the two bacteria and are in the process of characterizing them. A previous report of the failure to detect a homologous gene in P. haemolytica A1 by Southern hybridization using the H. somnus 31‐kDa antigen gene as a probe [10] could be due to the conditions used in the hybridization. Using plpD as the probe, we have detected hybridization signals in P. haemolytica A1 and H. somnus (data not shown) in agreement with the present sequence comparison.…”

Characterization of a fourth lipoprotein from Pasteurella haemolytica A1 and its homology to the OmpA family of outer membrane proteins

“…We have identi¢ed at least four other homologous antigen genes within the two bacteria and are in the process of characterizing them. A previous report of the failure to detect a homologous gene in P. haemolytica A1 by Southern hybridization using the H. somnus 31-kDa antigen gene as a probe [10] could be due to the conditions used in the hybridization. Using plpD as the probe, we have detected hybridization signals in P. haemolytica A1 and H. somnus (data not shown) in agreement with the present sequence comparison.…”

“…A homology search comparing Plp4 with sequences in GenBank showed signi¢cant homologies with several prokaryotic outer membrane proteins and lipoproteins. The greatest similarity was detected with a 31-kDa`hemolytic' antigen of H. somnus [10]. The amino acids of Plp4 and the H. somnus antigen are 63% identical and 13.9% similar for a total of 76.9% homology over 273 amino acids.…”

Immunology and Prevention of Infection in Feedlot Cattle

“…2l , 112, 114 It is likely that antibody against these potential virulence factors may help to protect the calf against H. somnus-induced pneumonia; however, there are scant data to support this hypothesis. In addition, there are two proteins that have been isolated from H. somnus, a 40-kD protein and a 31-kD protein, that have been implicated as important antigens for inducing immunity.3B, 113 The role of T-helper 1 cells or a secretory IgA response in protection from H. somnus-induced pneumonia has not been thoroughly investigated. There is evidence that gamma interferon, which can be produced during aT-helper 1 cell immune response, can help to protect the calf against H. somnus-induced pneumonia.…”