The cloning, expression and sequence analysis of a second Porphyromonas gingivalis gene that codes for a protein involved in hemagglutination

Abstract

It has been suggested that Porphyromonas gingivalis may possess more than one hemagglutinin. We have previously reported the cloning of a gene (hagA) that encodes a hemagglutinin. In this study we report the cloning, characterization, and sequencing of a second gene (hagB) that encodes a protein that also appears to be involved in hemagglutination. Antiserum to the clone (ST 7) was found to inhibit hemagglutination by P. gingivalis 381, and hemagglutinating inhibition activity of anti-P. gingivalis antiserum was reduced by adsorption of the antiserum with cells of clone ST 7. Restriction mapping and Southern analysis indicates there is little or no DNA homology between this cloned 4.8-kb HindIII DNA fragment and a cloned hemagglutinin gene we have previously described. Minicell analysis of the cloned P. gingivalis chromosomal DNA fragment revealed that the major gene product is a 49-kDa protein. Immunoaffinity chromatography using purified rabbit immunoglobulin G against the cloned protein resulted in the purification of a major reactive 49- to 50-kDa protein from a P. gingivalis cell lysate. Nucleotide sequence analysis revealed the hagB open reading frame to be 1053 nucleotides in length with a mol% G+C of 59.9% coding for a protein of 350 residues with a calculated molecular weight of 39.375 kDa. This protein was also determined to be basic and hydrophilic and to contain a potential signal peptide. Comparison of both the nucleotide and derived amino acid sequences with computer-based databases did not reveal any significant homologies between habB and any other previously sequenced genes.

Mechanisms of Monophosphoryl Lipid A Augmentation of Host Responses to Recombinant HagB fromPorphyromonas gingivalis

“…The fimbriae and hemagglutinins appear to be involved in the attachment of P. gingivalis to host tissues (11,22,32,51,61). A number of hemagglutinins have been identified and their genes have been cloned (16, 36-38, 50, 51).…”

Hemagglutinin B Is Involved in the Adherence of Porphyromonas gingivalis to Human Coronary Artery Endothelial Cells

“…Clindamycin was added to the media at 5 g/ml to maintain the HagB mutant of P. gingivalis 381. Escherichia coli JM109 containing pUC9 with or without a 4.8-kb DNA fragment (ST7) containing hagB was grown aerobically on Luria-Bertani (LB) plates or in LB broth (Difco) with 100 g/ml ampicillin, as described previously (49). For E. coli M15[pREP4]pQE-31 (QIAGEN Inc., Valencia, CA) and the HagB expression strain E. coli M15[pREP4]pQE-31-TX1, 100 g/ml ampicillin and 5 g/ml kanamycin were added to the media (33).…”

“…Hemagglutinins are also considered important virulence factors, as they can be a mechanism to acquire hemin, necessary for bacterial growth, from erythrocytes (39). Several hag genes, encoding hemagglutinins of P. gingivalis, have been previously described and cloned (20,40,48,49). However, the importance of the hemagglutinins in the colonization process of P. gingivalis remains to be determined.…”

Long-Term Immunological Memory Induced by Recombinant Oral Salmonella Vaccine Vectors

“…The hagB gene codes for a hemagglutinin from the periodontopathogen Porphyromonas gingivalis and is a potential virulence factor (15,19). We have previously shown that mice immunized intragastrically with Salmonella serovar Typhimurium expressing the hagB gene exhibit a vigorous serum immunoglobulin G (IgG) and IgA response to purified, recombinant HagB as well as a significant mucosal IgA response in saliva, gut secretions, and vaginal washes (5).…”