Prevalence of P.intermedia and P.nigrescens and preparation of their hemolytic toxins

2000 Fiscal Year Final Research Report Summary

• Project/Area Number: 11671816
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Morphological basic dentistry
• Research Institution: NIHON UNIVERSITY
• Principal Investigator: HIRASAWA Masatomo Nihon University, Microbiology, Associate professor, 松戸歯学部, 助教授 (60095453)
• Co-Investigator(Kenkyū-buntansha): TAKADA Kazuko Nihon University, Microbiology, Lecturer, 松戸歯学部, 講師 (20120496)
• Project Period (FY): 1999 – 2000
• Keywords: Periodontitis / Black-pigmented Gram negative rods / Prevotella intermedia / Prevotella nigrescens / Hemolytic toxin

Research Abstract

The purpose of this study was to determined the distribution of the putative periodontal pathogen Prevotella intermedia and Prevotella nigrescens in periodontal health and disease patients. Black-pigmented anaerobic Gram-negative rods (BPRs) were more numerous in samples from patients with periodontal disease than in those from healthy individuals. The BPRs were identified as P.intermedia and P.nigrescens. Sites exhibiting only P.intermedia or P.nigrescens were found in 47.1 % and 11.8 % of periodontal and 23.1 % and 38.5 % of healthy subjects, respectively. The remaining 41.2 % and 38.5 % possessed both P.intermedia and P.nigrescens, the former of which was predominant. These results suggested that P.intermedia may be associated with periodontal disease more than P.nigrescens.
The hemolysin from P.intermedia has been purified from culture supernatant and characterized. The hemolysin produced clear β-hemolytic zone on blood agar plate. The isolation and purification procedure involved a mmonium sulfate and polyethylene glycol precipitations and ion-exchange chromatographies on DEAE-Sephacel and CM-Sepharose. The optimal pH of the hemolysin was 7.5. Hemolytic activity was stimulated by reductants. Trypsin or heat treatment resulted in complete inhibition of hemolytic activity. Cation and EDTA did not affect the activity. These results indicate that the β-hemolysin from P.intermedia, appeared to belong to a family of toxins known as the thiol-activated toxin.
The other hemolytic substance was extracted and separated from culture supernatant by ammonium sulfate fractionation, Sephadex LH-20 and Wakogel C-200 column chromatographies. The hemolytic substance showed 0.97 Rf value on thin layer chromatography (TLC) using chloroform : methanol : water (65 : 35 : 5) as a developing solvent. Quantitative analysis of sugar ant TLC analysis showed that the hemolytic substance did not consist of choline, amino group, phosphate group and carbohydrate. The hemolytic stubstance was not affected by 60℃ for 30 minutes heating and trypsin treatment. The hemolytic substance may be regarded as simple lipid or derived lipid.