Molecular Genetics on Proteases of Porphyromonas

1996 Fiscal Year Final Research Report Summary

• Project/Area Number: 07457071
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Bacteriology (including Mycology)
• Research Institution: Nippon Dental University
• Principal Investigator: YOSHIKAWA Masanosuke Nippon Dental University, Department of Microbiology, Professor, 歯学部, 教授 (80012714)
• Co-Investigator(Kenkyū-buntansha): KUMAGAI Yumi Nippon Dental University, Department of Microbiology, assistant professor, 歯学部, 助手 (90277591) ; KURAMOCHI Toshiaki Nippon Dental University, Department of Microbiology, assistant professor, 歯学部, 助手 (80277590) ; TAKAHASHI Yukihiro Nippon Dental University, Department of Microbiology, lecturer, 歯学部, 講師 (00281436) ; KONISHI Kiyoshi Nippon Dental University, Department of Microbiology, associate professor, 歯学部, 助教授 (20178289)
• Project Period (FY): 1995 – 1996
• Keywords: protease / Porphyromonas / molecular genetics / virulence

Research Abstract

Porphyromonas gingivalis is one of the etiological pathogens in adult periodontitis. This organism produces several proteases that seem to be major virulence factors. In this project, we study the properties of proteases of P.gingivalis by protein chemical and molecular genetical approaches.
1. We prepared plasmid libraries containing the fragments of P.gingivalis genome. Escherichia coli transformed by these libraries, were screened for the degradation activity of casein. About 10,000 clones were screened, and only tpr gene encoding tpr protease was obtained consequently in our screening system. Another screening method will be performed for the cloning of additional protease gene of P.gingivalis.
2. Although Arg-gingipain is the best characterized protease among those of P.gingivalis, expression of mature protein in E.coli has not yet been reported. We established the expression system of Arg-gingipain. We will characterize this protease by use of site-directed mutagenesis method.
3. Glysylprolyl peptidase of P.gingivalis is thought to play an important role in degradation of collagen in the host tissues. This enzyme was purified completely, and several partial peptide sequences were determined with an automated peptide sequencer after lysyl endopeptidase-digestion. We performed PCR method using the oligonucleotide primers deduced from the peptide sequence data. About 1,500 base paired nucleotide was amplified, and the sequence data suggested that the peptide belongs to the group of eukaryotic dipeptidyl peptidase IV.A cloning of the gene encoding this peptidase was successfully carried out, and the determination of complete sequence of nucleotide is now in progress.