Functional analysis of Porphyromonas gingivalis HBP35 by amino acid substitution

Objectives: Periodontitis is one of the most common oral diseases resulting from the infection of Porphyromonas gingivalis. Our strategy to prevent this incidence is to establish passive immunization system where endogenous antibodies could directly be applied to neutralize virulent factors associated with this bacterium. We focused our attention on the P. gingivalis 35 kDa surface protein, or HBP35, since this protein is involved in not only the coaggregation with oral miroflora but also hemin-binding. In addition, nucleotide sequencing of the gene, hbp35 (Gene ID pg0554), coding for this protein revealed the presence of a total of four Cys residues. Of these, the first two consisted of the amino acid stretch, EVFTAEWCGYCPGGK, a catalytic center for thioredoxin. To prepare the effective antibody for passive immunization, functional analysis of this protein was carried out following amino acid substitution. Methods: To introduce the site-directed mutagenesis, we exploited a simple, rapid, and non-expensive approach based on the utilization of only the T4 DNA polymerase, and all four codons coding for Cys residues were successfully altered to those coding for Ser residues. The mutagenized plasmids were transformed into Escherichia coli disulfide bond formation-deficient strain, JCB 572, defective in alkaline phosphatase (AP) activity, and poor motility on soft agar plate due to inefficient S-S bond formation. Results: E. coli dsb mutant harboring the native hbp35 restored the AP activity while those possessing either first or second Cys to Ser-mutation diminished this complementation. Mutation in the third or fourth Cys was no effect on AP activity. On the other hand, the dsb transformants harboring the mutagenized individual plasmid exhibited rather complex motile activity profile on soft agar plate. Conclusion: The present results clearly indicated that, in addition to previously identified function, coaggregation and hemin-binding, the HBP35 could participate in disulfide bond formation in P. gingivalis cells.